Cyclopamine lactam analogs and methods of use thereof

ABSTRACT

The present invention relates to steroidal alkaloids useful in the treatment of hedgehog pathway related disorders, particularly cancer.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.13/102,395, filed May 6, 2011 now U.S. Pat. No. 8,293,760, which is adivisional of U.S. application Ser. No. 12/044,878, filed on Mar. 7,2008 now U.S. Pat. No. 7,964,590, which claims the benefit of priorityto U.S. Provisional Application Ser. No. 60/893,591, filed Mar. 7, 2007;each of these prior applications is incorporated herein by reference inits entirety.

TECHNICAL FIELD

The present invention generally relates to cyclopamine analogs,pharmaceutical compositions thereof, and methods for using such analogsand compositions. These compounds and compositions can be useful for thetreatment of hedgehog mediated disorders, such as cancer and psoriasis.

BACKGROUND ART

The Hedgehog polypeptide is a secreted protein that functions as asignaling ligand in the hedgehog pathway. Three different forms of thehedgehog protein are found in humans; Sonic hedgehog (Shh), Deserthedgehog (Dhh) and Indian hedgehog (Ihh) Sonic hedgehog is the mostprevalent hedgehog member in mammals and also is the best characterizedligand of the hedgehog family. Prior to secretion, Shh undergoes anintramolecular cleavage and lipid modification reaction. The lipidmodified peptide is responsible for signaling activities.

Inhibition of the hedgehog pathway in certain cancers has been shown toresult in inhibition of tumor growth. For example, anti-hedgehogantibodies have been shown to antagonize the function of the hedgehogpathway and inhibit the growth of tumors. Small molecule inhibition ofhedgehog pathway activity has also been shown to result in cell death ina number of cancer types.

Research in this area has focused primarily on the elucidation ofhedgehog pathway biology and the discovery of new hedgehog pathwayinhibitors. Although inhibitors of the hedgehog pathway have beenidentified, there still exists the need to identify more potentinhibitors of the hedgehog pathway.

SUMMARY

The present invention relates to analogs of steroidal alkaloids,pharmaceutical compositions, and methods of using them.

The invention includes compounds of Formula 1, compositions comprisingat least one such compound, and methods of using the compounds andcompositions, where Formula 1 is:

or a pharmaceutically acceptable salt thereof;wherein;

R¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, hydroxyl, aralkyl,heteroaryl, heteroaralkyl, haloalkyl, alkoxyl, —SR²⁰, —OR²⁰,—N(R²⁰)(R²⁰), —C(O)R²⁰, —CO₂R²⁰, —OC(O)R²⁰, —C(O)N(R²⁰)(R²⁰),—N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)(R²⁰)(R²⁰), —S(O)R²⁰, —S(O)₂R²⁰,—S(O)₂N(R²⁰)(R²⁰), —N(R²⁰)S(O)₂R²⁰, —[(W)—C(O)]_(p)R²⁰,—[(W)—C(O)O]_(p)R²⁰, —[(W)—OC(O)]_(p)R²⁰, —[(W)—SO₂]_(p)R²⁰,—[(W)—N(R²⁰)SO₂]_(p)R²⁰, —[(W)—C(O)N(R²⁰)]_(p)R²⁰, —[(W)—O]_(p)R²⁰,[(W)—N(R²⁰)]_(p)R²⁰ or —[(W)—S]_(p)R²⁰;

each of R², R⁷ and R¹³ is independently H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, alkoxyl, aryloxy, acyloxy,halide, hydroxyl, amino, alkylamino, arylamino, acylamino, aralkylamino,alkylseleno, aralkylseleno, arylseleno, alkylthio, aralkylthio,arylthio, heteroaryl, or heteroaralkyl;

R³ is H; or R² and R³ taken together form a bond;

each of R⁴ and R⁵ independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, nitrile, aralkyl, alkoxyl, aryloxy, acyloxy, halide,sulfhydryl, alkylthio, arylthio, aralkylthio, hydroxyl, amino,alkylamino, arylamino, acylamino, aralkylamino, heteroaryl, orheteroaralkyl;

or R⁴ and R⁵ taken together form ═O, ═S, ═N(R²⁰), ═N—OR²⁰ or═N(N(R²⁰)₂);

R⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, oraralkyl;

each of R⁸ and R¹² independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, heterocycloalkyl, or aralkyl; or R⁷ and R⁸ taken togetherform a bond; or R¹² and R¹³ taken together form a bond

each of R⁹ and R¹⁰ independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, nitrile, aralkyl, heteroaryl, or heteroaralkyl; or R⁹ andR¹⁰ taken together form ═O, ═N(R²⁰), ═N—OR²⁰, or ═S;

R¹¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, —C(O)R²⁰, —C(S)R²⁰, —CO₂R²⁰, —SO₂R²⁰, —C(O)N(R²⁰)(R²⁰), or—C(S)N(R²⁰)(R²⁰); or has the formula —[C(R²⁰)₂]_(q)—R²¹;

R²⁰ independently for each occurrence is H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl,or —[C(R)₂]_(q)—R²¹, where each R is independently H or C1-C6 alkyl; orany two occurrences of R²⁰ can be taken together to form a 4-8 memberedoptionally substituted ring which contains 0-3 heteroatoms selected fromN, O, S, and P;

R²¹ independently for each occurrence is H, cycloalkyl, aryl,heteroaryl, heterocyclyl; alkoxyl, aryloxy, acyloxy, halide, sulfhydryl,alkylthio, arylthio, aralkylthio, hydroxyl, amino, acylamino, amido, ora carbonyl-containing group;

R²² independently for each occurrence is H, halide, ester, amide, ornitrile;

p is 0, 1, 2, 3, 4, 5, or 6;

q is 0, 1, 2, 3, 4, 5, or 6;

W is a diradical;

X is a bond or —C(R²²)₂—

and each alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, whether alone or part of anothergroup, is optionally substituted.

In other aspects, the invention includes a compound of formula 15, aswell as compositions comprising at least one such compound and methodsof using such compounds and compositions for treatment of conditionssuch as hyperproliferative disorders, including cancer, that aremediated by the hedgehog pathway. The compounds of formula 15 arerepresented by:

A compound of formula 15:

or a pharmaceutically acceptable salt thereof;wherein;each of A and B independently is —N(R¹³)—, —(C═O)—, or —(C═S)—;R¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, hydroxyl, aralkyl,heteroaryl, heteroaralkyl, haloalkyl, alkoxyl, —SR²⁰, —OR²⁰,—N(R²⁰)(R²⁰), —C(O)R²⁰, —CO₂R²⁰, —OC(O)R²⁰, —C(O)N(R²⁰)(R²⁰),—N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)N(R²⁰)(R²⁰), —S(O)R²⁰, —S(O)₂R²⁰,—S(O)₂N(R²⁰)(R²⁰), —N(R²⁰)S(O)₂R²⁰, —[(W)—C(O)]_(p)R²⁰,—[(W)—C(O)O]_(p)R²⁰, —[(W)—OC(O)]_(p)R²⁰, —[(W)—SO₂]_(p)R²⁰,—[(W)—N(R²⁰)SO₂]_(p)R²⁰, —[(W)—C(O)N(R²⁰)]_(p)R²⁰, [(W)—O]_(p)R²⁰,—[(W)—N(R²⁰)]_(p)R²⁰, or —[(W)—S]_(p)R²⁰;each of R², R⁷ and R¹⁰ is independently H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, alkoxyl, aryloxy, acyloxy,halide, hydroxyl, amino, alkylamino, arylamino, acylamino, aralkylamino,alkylseleno, aralkylseleno, arylseleno, alkylthio, aralkylthio,arylthio, heteroaryl, or heteroaralkyl;R³ is H; or R² and R³ taken together form a bond;each of R⁴ and R⁵ independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, nitrile, aralkyl, alkoxyl, aryloxy, acyloxy, halide,sulfhydryl, alkylthio, arylthio, aralkylthio, hydroxyl, amino,alkylamino, arylamino, acylamino, aralkylamino, heteroaryl, orheteroaralkyl; or R⁴ and R⁵ taken together form ═O, ═S, ═N(R²⁰), ═N—OR²⁰or ═N(N(R²⁰)₂);R⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, oraralkyl; or R⁶ and R¹⁰ taken together form a bond;each of R¹¹, R¹², R¹⁴ and R¹⁵ a independently is H, alkyl, alkenyl,alkynyl, aryl, cycloalkyl, heterocycloalkyl, or aralkyl; or R¹¹ and R¹²taken together form a bond; or R⁷ and R¹⁴ taken together form a bond;R¹³ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, —C(O)R²⁰, —C(S)R²⁰, CO₂R²⁰, —SO₂R²⁰, —C(O)N(R²⁰)(R²⁰), or hasthe formula —C(S)N(R²⁰)(R²⁰); or has the formula —[C(R²⁰)₂]—R²¹;

R²⁰ independently for each occurrence is H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl,or —[C(R)₂]_(q)—R²¹, where each R is independently H or C1-C6 alkyl; orany two occurrences of R²⁰ can be taken together to form a 4-8 memberedoptionally substituted ring which contains 0-3 heteroatoms selected fromN, O, S, and P;

R²¹ independently for each occurrence is H, cycloalkyl, aryl,heteroaryl, heterocyclyl; alkoxyl, aryloxy, acyloxy, halide, sulfhydryl,alkylthio, arylthio, aralkylthio, hydroxyl, amino, acylamino, amido, ora carbonyl-containing group;

R²² independently for each occurrence is H, halide, ester, amide, ornitrile;

p is 0, 1, 2, 3, 4, 5, or 6;

q is 0, 1, 2, 3, 4, 5, or 6;

W is a diradical;

X is a bond or —C(R²²)₂—;

and each alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, whether alone or part of anothergroup, is optionally substituted; provided that when A is —N(R¹³)—; Bmust be —(C═O)—, or —(C═S)—; and

provided that when A is —(C═O)—, or —(C═S)—; B must be —N(R¹³)—.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts the carboskeleton of a steroidal alkaloid with the ringslabeled A-F.

MODES OF CARRYING OUT THE INVENTION Definitions

The definitions of terms used herein are meant to incorporate thepresent state-of-the-art definitions recognized for each term in thechemical and pharmaceutical fields. Where appropriate, exemplificationis provided. The definitions apply to the terms as they are usedthroughout this specification, unless otherwise limited in specificinstances, either individually or as part of a larger group.

As used herein, the definition of each expression, e.g., alkyl, m, n,etc., when it occurs more than once in any structure, is intended to beindependent of its definition elsewhere in the same structure.

The term “acyl” as used herein refers to a group of the general formulaR—C(═O)—, where R can be H, alkyl, aryl, or aralkyl. In typical acylgroups, R is H or C1-C6 alkyl, which is optionally substituted, or R canbe aralkyl, wherein the aryl portion of the aralkyl is a 5-7 memberedaromatic or heteroaromatic ring, and the alkyl portion is a C1-C4alkylene group; and both the alkyl and aryl portions are optionallysubstituted as described herein for such groups. Benzyl,p-methoxybenzyl, and phenylethyl are examples of a typical aralkyl.

The term “acylamino” refers to a moiety that may be represented by thegeneral formula:

wherein R50 is as defined below, and R54 represents a hydrogen, analkyl, an alkenyl or —(CH₂)_(m)—R61, where m and R61 are as definedbelow.

The terms “alkenyl” and “alkynyl” refer to unsaturated aliphatic groupsanalogous in length and possible substitution to the alkyls describedbelow, but that contain at least one double or triple bond respectively.Alkenyl and alkynyl groups may be substituted with the same groups thatare suitable as substituents on alkyl groups, to the extent permitted bythe available valences. Typical alkenyl and alkynyl groups contain 2-10carbons in the backbone structure.

The terms “alkoxyl” or “alkoxy” refers to an alkyl group, as definedbelow, having an oxygen radical attached thereto. Representative alkoxylgroups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. Thealkyl portion of an alkoxy group is sized like the alkyl groups, and canbe substituted by the same groups that are suitable as substituents onalkyl groups, to the extent permitted by the available valences.

The term “alkyl” refers to the radical of saturated aliphatic groups,including straight-chain alkyl groups, branched-chain alkyl groups,cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, andcycloalkyl substituted alkyl groups. In certain embodiments, a straightchain or branched chain alkyl has 30 or fewer carbon atoms in itsbackbone (e.g., C₁-C₃₀ for straight chain, C₃-C₃₀ for branched chain),20 or fewer. Typically, an alkyl group contains 1-10 carbon atoms as itsbackbone, and may be substituted or unsubstituted. Likewise, certaincycloalkyls have from 3-10 carbon atoms in their ring structure, andothers have 5, 6 or 7 carbons in the ring structure. Unless otherwiseindicated, alkyl and cycloalkyl groups, whether alone or as part ofanother group such as an aralkyl group, can be substituted by suitablesubstituents such as, but not limited to, halogen, azide, oxo, acyl,cycloalkyl, hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, oximino,amido, acylamino, phosphonate, phosphinate, carbonyl, carboxylic acidsor their esters or amides, silyl, alkoxy, alkylthio, alkylsulfonyl,alkylsulfonamido, ketone, aldehyde, ester, heterocyclyl, aromatic orheteroaromatic moieties, —CF₃, —CN, and the like.

Where alkyl, alkenyl, or alkynyl is part of another group, such as inalkoxy, alkylthio, etc., or it is a substituent on another group, it isfrequently an optionally substituted lower alkyl group or lower alkenylgroup, or lower alkynyl group having up to six carbon atoms. For suchpurposes, the typical substituents include halo, —OR′, —SR′, —SO₂R′,—SO₂NR′₂, COOR′, CONR′₂, oxo, —NR′₂, NR′C(O)R′, NR′C(O)OR′, NR′SO₂R′,OC(O)R′, where each R′ is independently H or unsubstituted C₁-C₆ alkyl,C₂-C₆ alkenyl, or C₂-C₆ alkynyl.

The term “alkylthio” refers to an alkyl group, as defined above, havinga sulfur radical attached thereto. In certain embodiments, the“alkylthio” moiety is represented by one of —S-alkyl, —S-alkenyl,—S-alkynyl, and —S—(CH₂)_(m)—R61, wherein m and R61 are defined below.Representative alkylthio groups include methylthio, ethyl thio, and thelike.

The term “amido” is art recognized as an amino-substituted carbonyl andincludes a moiety that may be represented by the general formula:

wherein R50 and R51 are as defined below. Certain embodiments of theamide in the present invention will not include imides which may beunstable.

The terms “amine” and “amino” are art-recognized and refer to bothunsubstituted and substituted amines, e.g., a moiety that may berepresented by the general formulas:

wherein R50, R51 and R52 and R53 each independently represent ahydrogen, an alkyl, an alkenyl, —(CH₂)_(m)—R61, or R50 and R51 (or R50and R52 in the ammonium species), taken together with the N atom towhich they are attached complete a heterocycle having from 4 to 8 atomsin the ring structure; R61 represents an aryl, a cycloalkyl, acycloalkenyl, a heterocycle or a polycycle; and m is zero or an integerin the range of 1 to 8. In other embodiments, R50 and R51 (andoptionally R52) each independently represent a hydrogen, an alkyl, analkenyl, or —(CH₂)_(m)—R61. Thus, the term “alkylamine” includes anamine group, as defined above, having a substituted or unsubstitutedalkyl attached thereto, i.e., at least one of R50 and R51 is an alkylgroup.

The term “aralkyl”, as used herein, whether alone or as part of a groupname such as, for example, aralkyloxy, refers to an alkyl group asdescribed herein substituted with an aryl group as described herein(e.g., an aromatic or heteroaromatic group). Both the alkyl and the arylportion of each aralkyl group are typically optionally substituted.Typical aralkyl groups include, for example, groups of general formulaAr—(CH₂)_(t)—, where Ar represents an aryl ring and t is an integer from1-6.

The term “aryl” as used herein, whether alone or as part of another namesuch as ‘aryloxy’, includes 5-, 6- and 7-membered single-ring aromaticgroups that may include from zero to four heteroatoms selected from N, Oand S as ring members, as well as fused bicyclic an tricyclic systemsconsisting of such rings, for example, benzene, anthracene, naphthalene,pyrene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole,triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, andthe like. Those aryl groups having heteroatoms in the ring structure mayalso be referred to as “aryl heterocycles” or “heteroaromatics.” Thearomatic ring may be substituted as available valences permit at one ormore ring positions with such substituents as described above, forexample, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, alkoxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carbonyl-containing group, silyl, ether,alkylthio, sulfonyl, sulfonamido, ketone, aldehyde, ester, heterocyclyl,aromatic or heteroaromatic moieties, —CF₃, —CN, or the like. The term“aryl” also includes polycyclic ring systems having two or more cyclicrings, often two or three rings, in which two or more carbons are commonto two adjoining rings (the rings are “fused rings”) wherein at leastone of the rings is aromatic, e.g., the other cyclic rings may becycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.In some embodiments, each aryl is selected from phenyl, thiophene,furan, pyrrole, pyridine, pyrimidine, pyrazole, imidazole, oxazole,thiazole, isoxazole and isothiazole. Phenyl is sometimes preferred.

The term “Brønsted acid” refers to any substance that can act as ahydrogen ion (proton) donor.

The term “carbonyl-containing group” includes such moieties as may berepresented by the general formulas:

wherein X50 is a bond or represents an oxygen or a sulfur, and each ofR55 and R56 represents independently a hydrogen, an alkyl, an alkenyl,—(CH₂)_(m)—R61 or a cation representing a pharmaceutically acceptablesalt, where m and R61 are defined above. In some embodiments where acarbonyl-containing group is present, it is a carboxylic acid or ester,or an acyloxy group; X50 is O in such embodiments, and R55 or R56,whichever is present, is often H or an optionally substituted alkylgroup.

The term “diradical” refers to any of a series of divalent groups fromalkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, aralkyl,heteroaryl, and heteroaralkyl groups, each of which can be optionallysubstituted. For example,

is an alkyl diradical;

is also an alkyl diradical;

is an aralkyl diradical; and

is an (alkyl)heteroaralkyl diradical. Typical examples include alkylenesof general structure (CH₂)_(x) where x is 1-6, and correspondingalkenylene and alkynylene linkers having 2-6 carbon atoms and containingone or more double or triple bonds; cycloalkylene groups having 3-8 ringmembers; groups such as (CH₂)_(a)C(═O)(CH₂)_(b), where a and b are eachintegers from 0-4; and aralkyl groups wherein one open valence is on thearyl ring and one is on the alkyl portion such as

and its isomers. The alkyl, alkenyl, alkynyl, aryl, cycloalkyl,heterocycloalkyl, aralkyl, heteroaryl, and heteroaralkyl portions of adiradical are optionally substituted as described above.

The term “haloalkyl”, as used herein, refers to an alkyl group whereanywhere from 1 to all hydrogens have been replaced with a halide. A“perhaloalkyl” is where all of the hydrogens have been replaced with ahalide.

The term “heteroatom” as used herein means an atom of any element otherthan carbon or hydrogen. Examples of heteroatoms include boron,nitrogen, oxygen, phosphorus, sulfur and selenium. Typically, theheteroatoms are selected from N, O and S.

The term ‘heteroalkyl’ and ‘heterocycloalkyl’ refer to alkyl andcycloalkyl groups as described herein, wherein at least one carbon atomof the alkyl or cycloalkyl portion is replaced by a heteroatom selectedfrom N, O and S. Typical examples include methoxymethyl, allylthioethyl,dimethylaminoethyl, and tetrahydrofuranyl.

The terms “heterocyclyl” or “heterocyclic group” refer to 3- to10-membered ring structures, in some instances from 3- to 7-memberedrings, whose ring structures include at least one carbon atom and one tofour heteroatoms. Heterocycles can also be polycycles. Heterocyclylgroups include, for example, thiophene, thianthrene, furan, pyran,isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole,pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine,pyridazine, indolizine, isoindole, indole, indazole, purine,quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine,quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline,phenanthridine, acridine, pyrimidine, phenanthroline, phenazine,phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane,thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactamssuch as azetidinones and pyrrolidinones, sultams, sultones, and thelike. The heterocyclic ring may be substituted at one or more positionswith such substituents as described above, as for example, halogen,alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro,sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl,carbonyl-containing group, silyl, ether, alkylthio, sulfonyl, ketone,aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety,—CF₃, —CN, or the like.

The term “Lewis acid” refers to any substance that can act as anelectron pair acceptor.

Unless the number of carbons is otherwise specified, “lower alkyl” asused herein means an alkyl group, as defined above, but having from oneto ten carbons, in some embodiments from one to six carbon atoms in itsbackbone structure. Likewise, “lower alkenyl” and “lower alkynyl” havesimilar chain lengths. Certain alkyl groups are lower alkyls. In someembodiments, a substituent designated herein as alkyl is a lower alkyl.

As used herein, the term “nitro” means —NO₂; the term “halogen”designates —F, —Cl, —Br or —I; the term “sulfhydryl” means —SH; the term“hydroxyl” means —OH; and the term “sulfonyl” means —SO₂—.

The term “optionally substituted” as used herein indicates that aspecified group may be unsubstituted or it may be substituted with oneor more substituents to the extent consistent with the number ofavailable valences on the specified group. In some embodiments, eachoptionally substituted group is substituted with up to four substituentsor with 0-3 substituents.

The term “oxo” refers to a carbonyl oxygen

The terms “polycyclyl” or “polycyclic group” refer to two or more rings(e.g., cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/orheterocyclyls) in which two or more carbons are common to two adjoiningrings, e.g., the rings are “fused rings”. Rings that are joined throughnon-adjacent atoms are termed “bridged” rings. Each of the rings of thepolycycle may be substituted with such substituents as described above,as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl,hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate,phosphinate, carbonyl, carbonyl-containing group, silyl, ether,alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, anaromatic or heteroaromatic moiety, —CF₃, —CN, or the like.

The phrase “protecting group” as used herein means temporarysubstituents which protect a potentially reactive functional group fromundesired chemical transformations. Examples of such protecting groupsinclude esters of carboxylic acids, silyl ethers of alcohols, andacetals and ketals of aldehydes and ketones, respectively. The field ofprotecting group chemistry has been reviewed (Greene, T. W.; Wuts, P. G.M., Protective Groups in Organic Synthesis, 2^(nd) ed.; Wiley: New York,1991).

The term “sugar” as used herein refers to a natural or an unnaturalmonosaccharide, disaccharide or oligosaccharide comprising one or morepyranose and/or furanose rings. The sugar may be covalently bonded tothe steroidal alkaloid of the present invention through an ether linkageor through an alkyl linkage. In certain embodiments the saccharidemoiety may be covalently bonded to a steroidal alkaloid of the presentinvention at an anomeric center of a saccharide ring. Sugars mayinclude, but are not limited to ribose, arabinose, xylose, lyxose,allose, altrose, glucose, mannose, gulose, idose, galactose, talose,glucose, and trehalose.

The terms “triflyl”, “tosyl”, “mesyl”, and “nonaflyl” refer totrifluoromethanesulfonyl, p-toluenesulfonyl, methanesulfonyl, andnonafluorobutanesulfonyl groups, respectively. The terms “triflate”,“tosylate”, “mesylate”, and “nonaflate” refer totrifluoromethanesulfonate ester, p-toluenesulfonate ester,methanesulfonate ester, and nonafluorobutanesulfonate ester functionalgroups and molecules that contain said groups, respectively.

The term “thioxo” refers to a carbonyl sulfur (═S).

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms represent methyl, ethyl,phenyl, trifluoromethanesulfonyl, nonafluorobutanesulfonyl,p-toluenesulfonyl and methanesulfonyl, respectively. A morecomprehensive list of the abbreviations utilized by organic chemists ofordinary skill in the art appears in the first issue of each volume ofthe Journal of Organic Chemistry; this list is typically presented in atable entitled Standard List of Abbreviations.

It will be understood that “substitution” or “substituted with” includesthe implicit proviso that such substitution is in accordance withpermitted valence of the substituted atom and the substituent, and thatthe substitution results in a stable compound, e.g., which does notspontaneously undergo transformation such as by rearrangement,cyclization, elimination, etc.

Where two groups are “taken together form a bond,” if the groups areattached to atoms that are not otherwise directly bonded to each other,they represent a bond between the atoms to which they are attached. Ifthe groups are on atoms that are directly bonded to each other, theyrepresent an additional bond between those two atoms. Thus, for example,when R² and R³ taken together form a bond, the structure —C(A)R²—C(B)R³—represents —C(A)═C(B)—.

The invention, in one aspect, includes compounds of Formula 1:

and the pharmaceutically acceptable salt thereof, wherein:R¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, hydroxyl, aralkyl,heteroaryl, heteroaralkyl, haloalkyl, alkoxyl, —SR²⁰, —OR²⁰,—N(R²⁰)(R²⁰), —C(O)R²⁰, —CO₂R²⁰, —OC(O)R²⁰, —C(O)N(R²⁰)(R²⁰),—N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)N(R²⁰)(R²⁰), —S(O)R²⁰, —S(O)₂R²⁰,—S(O)₂N(R²⁰)(R²⁰), —N(R²⁰)S(O)₂R²⁰, —[(W)—C(O)]_(p)R²⁰,—[(W)—C(O)O]_(p)R²⁰, —[(W)—OC(O)_(p)]R²⁰, —[(W)—SO₂]_(p)R²⁰,—[(W)—N(R²⁰)SO₂]_(p)R²⁰, —[(W)—C(O)N(R²⁰)]_(p)R²⁰, —[(W)—O]_(p)R²⁰,—[(W)—N(R²⁰)]_(p)R²⁰, or —[(W)—S]_(p)R²⁰;each of R², R⁷ and R¹³ is independently H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, alkoxyl, aryloxy, acyloxy,halide, hydroxyl, amino, alkylamino, arylamino, acylamino, aralkylamino,alkylseleno, aralkylseleno, arylseleno, alkylthio, aralkylthio,arylthio, heteroaryl, or heteroaralkyl;R³ is H; or R² and R³ taken together form a bond;each of R⁴ and R⁵ independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, nitrile, aralkyl, alkoxyl, aryloxy, acyloxy, halide,sulfhydryl, alkylthio, arylthio, aralkylthio, hydroxyl, amino,alkylamino, arylamino, acylamino, aralkylamino, heteroaryl, orheteroaralkyl; or R⁴ and R⁵ taken together form ═O, ═S, ═N(R²⁰), ═N—OR²⁰or ═N(N(R²⁰)₂);R⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, oraralkyl;each of R⁸ and R¹² independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, heterocycloalkyl, or aralkyl; or R⁷ and R⁸ taken togetherform a bond; or R¹² and R¹³ taken together form a bondeach of R⁹ and R¹⁰ independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, nitrile, aralkyl, heteroaryl, or heteroaralkyl; or R⁹ andR¹⁰ taken together form ═O, ═N(R²⁰), ═N—OR²⁰, or ═S;R¹¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, —C(O)R²⁰, —C(S)R²⁰, —CO₂R²⁰, —SO₂R²⁰, —C(O)N(R²⁰)(R²⁰), or—C(S)N(R²⁰)(R²⁰); or has the formula —[C(R²⁰)₂]_(q)—R²¹;

R²⁰ independently for each occurrence is H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl,or —[C(R)₂]_(q)—R²¹, where each R is independently H or C1-C6 alkyl; orany two occurrences of R²⁰ can be taken together to form a 4-8 memberedoptionally substituted ring which contains 0-3 heteroatoms selected fromN, O, S, and P;

R²¹ independently for each occurrence is H, cycloalkyl, aryl,heteroaryl, heterocyclyl; alkoxyl, aryloxy, acyloxy, halide, sulfhydryl,alkylthio, arylthio, aralkylthio, hydroxyl, amino, acylamino, amido, orcarbonyl-containing group;

R²² independently for each occurrence is H, halide, ester, amide, ornitrile;

p is 0, 1, 2, 3, 4, 5, or 6;

q is 0, 1, 2, 3, 4, 5, or 6;

W is a diradical; and

X is a bond or —C(R²²)₂—;

and each alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, whether alone or part of anothergroup, is optionally substituted.

Certain compounds of the present invention may exist in particulargeometric or stereoisomeric forms. The present invention contemplatesall such compounds, including cis- and trans-isomers, R- andS-enantiomers, diastereomers, (D)-isomers, (L)-isomers, the racemicmixtures thereof, and other mixtures thereof, as falling within thescope of the invention. Additional asymmetric carbon atoms may bepresent in a substituent such as an alkyl group. All such isomers, aswell as mixtures thereof, are intended to be included in this invention.Where tautomers are possible in a compound of the invention, theinvention includes each tautomeric form. Where stereochemistry of achiral center is not expressly depicted or described, the structureincludes each isomer at that center. Where the absolute stereochemistryof a compound is depicted in a drawing of a structure, the depictedisomer is a preferred embodiment; a racemic mixture of each specificallydepicted compound is also an embodiment of the invention.

In some embodiments, the invention provides a compound of formula 1,wherein each of R⁷, R⁸, R¹² and R¹³ represents H. In some embodiments,R¹¹ is H or optionally substituted C1-C6 alkyl.

In some of the foregoing embodiments of the compounds of formula 1, R⁴and R⁵ are both H; or R⁴ and R⁵ taken together form ═O.

In some of the foregoing embodiments, R² and R³ taken together form abond, so the D-ring contains a double bond. In such embodiments, X issometimes a bond and it is sometimes CH₂. In some embodiments, R² and R³are each H.

In some of the foregoing embodiments, R⁹ and R¹⁰ are each H; in others,R⁹ and R¹⁰ taken together form ═O or ═S, so that the A-ring is a lactamor thiolactam. In some embodiments, R⁶ is H or Me.

In some of the foregoing embodiments, R¹ is preferably H or anoptionally substituted C1-C6 alkyl or aryl-(C1-C6)-alkyl. In other ofthe foregoing embodiments, R¹ is preferably of the form C(O)R²⁰, SO₂R²⁰or CO₂R²⁰, where R²⁰ is an optionally substituted C1-C6 alkyl oraryl-(C1-C6)-alkyl. In certain embodiments, when R¹ is COOR²⁰, R²⁰ isbenzyl, methyl, ethyl, or tert-butyl.

In some of the foregoing embodiments, R¹¹ is preferably H or anoptionally substituted C1-C6 alkyl or aryl-(C1-C6)-alkyl. In other ofthe foregoing embodiments, R¹¹ is preferably of the form C(O)R²⁰, SO₂R²⁰or CO₂R²⁰, where R²⁰ is an optionally substituted C1-C6 alkyl oraryl-(C1-C6)-alkyl. In certain embodiments, when R¹ is COOR²⁰, R²⁰ isbenzyl, methyl, ethyl, or tert-butyl.

In some of the foregoing embodiments, R⁷ and R⁸ are both H; in otherembodiments, when R⁷ and R⁸ are not H, R⁷ and R⁸ taken together form abond, so the A-ring contains a double bond.

In some of the foregoing embodiments, p is 0 or 1 independently at eachoccurrence. In some such embodiments, p is 1.

In some embodiments, the compound of Formula 1 is a compound of formula9:

or a pharmaceutically acceptable salt thereof;wherein;R¹, R⁴, R⁵, and R⁶ are as defined above for formula 1,each of R⁹ and R¹⁰ independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, nitrile, aralkyl, heteroaryl, or heteroaralkyl; or R⁹ andR¹⁰ taken together form ═O, ═N(R²⁰), ═N—OR²⁰, or ═S;R¹¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, —C(O)R²⁰, —C(S)R²⁰, —CO₂R²⁰, —SO₂R²⁰, —C(O)N(R²⁰)(R²⁰), or—C(S)N(R²⁰)(R²⁰); or has the formula —[C(R²⁰)₂]_(q)—R²¹;R²⁰ independently for each occurrence is H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl,or —[C(R)₂]_(q)—R²¹, where each R is independently H or C1-C6 alkyl; orany two occurrences of R²⁰ can be taken together to form a 4-8 memberedoptionally substituted ring which contains 0-3 heteroatoms selected fromN, O, S, and P;R²¹ independently for each occurrence is H, cycloalkyl, aryl,heteroaryl, heterocyclyl; alkoxyl, aryloxy, acyloxy, halide, sulfhydryl,alkylthio, arylthio, aralkylthio, hydroxyl, amino, acylamino, amido, orcarbonyl-containing group; andp is 0, 1, 2, 3, 4, 5, or 6;q is 0, 1, 2, 3, 4, 5, or 6;W is a diradical;X is a bond or —CH₂—;

and each alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, whether alone or part of anothergroup, is optionally substituted.

In some embodiments of the compound of formula 9, X is —CH₂—. In otherembodiments, X is a bond.

In some of the foregoing embodiments of compounds of formula 9, R⁶ is Hor optionally substituted C1-C6 alkyl. Sometimes R⁶ is Me.

In some embodiments of the compounds of formula 9, R¹ is H. In otherembodiments, R¹ is alkyl, alkenyl, alkynyl, aryl, cycloalkyl, hydroxyl,aralkyl, heteroaryl, heteroaralkyl, haloalkyl, alkoxyl,—C(O)N(R²⁰)(R²⁰), COOR²⁰, —[C(R²⁰)₂]_(p)—R²⁰, —[(W)—N(R²⁰)C(O)]_(p)R²⁰,—[(W)—C(O)]_(p)R²⁰, —[(W)—C(O)O]_(p)R²⁰, —[(W)—OC(O)]_(p)R²⁰,—[(W)—SO₂]_(p)R²⁰, —[(W)—N(R²⁰)SO₂]_(p)R²⁰, —[(W)—C(O)N(R²⁰)]_(p)R²⁰,—[(W)—O]_(p)R²⁰, —[(W)—N(R²⁰)]_(p)R²⁰, or —[(W)—S]_(p)R²⁰. Often R¹ isH, optionally substituted C1-C6 alkyl, C(O)R²⁰, SO₂R²⁰, or it is COOR²⁰.In certain embodiments, R¹ is H or C(O)R²⁰ or COOR²⁰, where R²⁰ isbenzyl, methyl, ethyl, or tert-butyl. In some of the foregoingembodiments, p is 0 or 1 independently at each occurrence. In some suchembodiments, p is 1.

In some embodiments of the compounds of formula 9, R⁴ and R⁵ are both H.In other such embodiments, R⁴ and R⁵ taken together form a bond.

In some embodiments of the compounds of formula 9, R⁹ and R¹⁰ takentogether form ═O or ═S. In many such embodiments, they are takentogether to form ═O.

In some embodiments, the compounds of formula 9 is selected from:

and the pharmaceutically acceptable salts of these compounds.

In another aspect, the invention provides a compound of formula 15:

or a pharmaceutically acceptable salt thereof, wherein;each of A and B independently is —N(R¹³)—, —(C═O)—, or —(C═S)—;R¹ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, hydroxyl, aralkyl,heteroaryl, heteroaralkyl, haloalkyl, alkoxyl, —SR²⁰, —OR²⁰,—N(R²⁰)(R²⁰), —C(O)R²⁰, —CO₂R²⁰, —OC(O)R²⁰, —C(O)N(R²⁰)(R²⁰),—N(R²⁰)C(O)R²⁰, —N(R²⁰)C(O)N(R²⁰)(R²⁰), —S(O)R²⁰, —S(O)₂R²⁰,—S(O)₂N(R²⁰)(R²⁰), —N(R²⁰)S(O)₂R²⁰, —[(W)—C(O)]_(p)R²⁰,—[(W)—C(O)O]_(p)R²⁰, —[(W)—OC(O)]_(p)R²⁰, —[(W)—SO₂]_(p)R²⁰,—[(W)—N(R²⁰)SO₂]_(p)R²⁰, —[(W)—C(O)N(R²⁰)]_(p)R²⁰, —[(W)—O]_(p)R²⁰,[(W)—N(R²⁰)]_(p)R²⁰, or —[(W)—S]_(p)R₂₀;each of R², R⁷ and R¹⁰ is independently H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, alkoxyl, aryloxy, acyloxy,halide, hydroxyl, amino, alkylamino, arylamino, acylamino, aralkylamino,alkylseleno, aralkylseleno, arylseleno, alkylthio, aralkylthio,arylthio, heteroaryl, or heteroaralkyl;R³ is H; or R² and R³ taken together form a bond;each of R⁴ and R⁵ independently is H, alkyl, alkenyl, alkynyl, aryl,cycloalkyl, nitrile, aralkyl, alkoxyl, aryloxy, acyloxy, halide,sulfhydryl, alkylthio, arylthio, aralkylthio, hydroxyl, amino,alkylamino, arylamino, acylamino, aralkylamino, heteroaryl, orheteroaralkyl; or R⁴ and R⁵ taken together form ═O, ═S, ═N(R²⁰), ═N—OR²⁰or ═N(N(R²⁰)₂);R⁶ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl, oraralkyl; or R⁶ and R¹⁰ taken together form a bond;each of R¹¹, R¹², R¹⁴ and R¹⁵ independently is H, alkyl, alkenyl,alkynyl, aryl, cycloalkyl, heterocycloalkyl, or aralkyl; or R¹¹ and R¹²taken together form a bond; or R⁷ and R¹⁴ taken together form a bond;R¹³ is H, alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, —C(O)R²⁰, —C(S)R²⁰, —CO₂R²⁰, —SO₂R²⁰, —C(O)N(R²⁰)(R²⁰); or—C(S)N(R²⁰)(R²⁰); or has the formula —[C(R²⁰)₂]_(q)—R²¹;

R²⁰ independently for each occurrence is H, alkyl, alkenyl, alkynyl,aryl, cycloalkyl, heterocycloalkyl, aralkyl, heteroaryl, heteroaralkyl,or —[C(R)₂]_(q)—R²¹, where each R is independently H or C1-C6 alkyl; orany two occurrences of R²⁰ can be taken together to form a 4-8 memberedoptionally substituted ring which contains 0-3 heteroatoms selected fromN, O, S, and P;

R²¹ independently for each occurrence is H, cycloalkyl, aryl,heteroaryl, heterocyclyl; alkoxyl, aryloxy, acyloxy, halide, sulfhydryl,alkylthio, arylthio, aralkylthio, hydroxyl, amino, acylamino, amido, orcarbonyl-containing group;

R²² independently for each occurrence is H, halide, ester, amide, ornitrile;

p is 0, 1, 2, 3, 4, 5, or 6;

q is 0, 1, 2, 3, 4, 5, or 6;

W is a diradical;

X is a bond or —C(R²²)₂—;

and each alkyl, alkenyl, alkynyl, aryl, cycloalkyl, heterocycloalkyl,aralkyl, heteroaryl, heteroaralkyl, whether alone or part of anothergroup, is optionally substituted;

provided that when A is —N(R¹³)—; B must be —(C═O)—, or —(C═S)—; and

provided that when A is —(C═O)—, or —(C═S)—; B must be —N(R¹³)—.

In some embodiments of the compounds of formula 15, A is —N(R¹³)— and Bis C═O. In other embodiments of the compounds of formula 15, B is—N(R¹³)— and A is C═O.

In some of the foregoing embodiments of the compounds of formula 15, R¹³is H. In other such embodiments, R¹³ is COOR²⁰ or SO₂R²⁰, wherein R²⁰ isC1-C6 alkyl or aryl-(C1-C6)-alkyl, and each alkyl and aryl is optionallysubstituted. In certain embodiments, when R¹³ is COOR²⁰, R²⁰ is benzyl,methyl, ethyl, or tert-butyl.

In some of the foregoing embodiments of the compounds of formula 15, R¹¹and R¹² are each H. In others, R¹¹ and R¹² are taken together to form abond.

In some of the foregoing embodiments of the compounds of formula 15, R²and R³ taken together form a bond. In other embodiments, R² and R³ areboth H.

In some of the foregoing embodiments of the compounds of formula 15, R⁴and R⁵ are each H; in other such embodiments, R⁴ and R⁵ taken togetherform ═O.

In some of the foregoing embodiments of the compounds of formula 15, R⁷and R¹⁰ are each H. In some of the foregoing embodiments of thecompounds of formula 15, when R⁷ is not H, R⁷ and R¹⁴ are taken togetherto form a bond. In other embodiments, when R¹⁰ is not H, R¹⁰ and R⁶ aresometimes taken together to form a bond.

In some of the foregoing embodiments of the compounds of formula 15, R¹is H. In other such embodiments, R¹ is COOR²⁰ or SO₂R²⁰, wherein R²⁰ isC1-C6 alkyl or aryl-(C1-C6)-alkyl, and each alkyl and aryl is optionallysubstituted. In certain embodiments, when R¹ is COOR²⁰, R²⁰ is benzyl,methyl, ethyl, or tert-butyl.

In some of the foregoing embodiments, the compound of formula 15, X isCH₂. In other such embodiments, X is a bond.

In some of the foregoing embodiments, p is 0 or 1 independently at eachoccurrence. In some such embodiments, p is 1.

In some embodiments, the compound of formula 15 is a compound of formula21:

where R¹, R⁴, R⁵, R⁷, R¹⁴, R¹³, R⁶, R²⁰, and X are as defined above forformula 15, and Y is O or S; or a pharmaceutically acceptable saltthereof.

In some embodiments of the compound of formula 21, X is —CH₂—. In otherembodiments, X is a bond.

In some of the foregoing embodiments of the compound of formula 21, Y isO. In some such embodiments, R⁶ is H.

In some of the foregoing embodiments of the compounds of formula 21, R¹is H. In other such embodiments, R¹ is COOR²⁰ or SO₂R²⁰, wherein R²⁰ isC1-C6 alkyl or aryl-(C1-C6)-alkyl, and each alkyl and aryl is optionallysubstituted. In certain embodiments, when R¹ is COOR²⁰, R²⁰ is benzyl,methyl, ethyl, or tert-butyl.

In some of the foregoing embodiments of the compounds of formula 21, R⁷and R¹⁴ are both H. In other such embodiments, R⁷ and R¹⁴ are takentogether to form a bond.

In some of the foregoing embodiments of the compound of formula 21, R⁴and R⁵ are both H. In other such embodiments, R⁴ and R⁵ are takentogether to form ═O.

In some of the foregoing embodiments of the compound of formula 21, R¹³is H or C1-C6 alkyl, such as methyl. In other such embodiments, R¹³ isCOOR²⁰ or SO₂R²⁰, wherein R²⁰ is C1-C6 alkyl or aryl-(C1-C6)-alkyl, andeach alkyl and aryl is optionally substituted. In certain embodiments,when R¹³ is COOR²⁰, R²⁰ is benzyl, methyl, ethyl, or tert-butyl. In someof the foregoing embodiments, p is 0 or 1 independently at eachoccurrence. In some such embodiments, p is 1.

In some of the foregoing embodiments, the compound of formula 21 hasformula 23:

wherein R¹, R¹³ and X are as defined for formula 15, and Y is O or S; ora pharmaceutically acceptable salt thereof.

In some embodiments of the compounds of formula 23, R¹ is H. In othersuch embodiments, R¹ is COOR²⁰ or SO₂R²⁰, wherein R²⁰ is C1-C6 alkyl oraryl-(C1-C6)-alkyl, and each alkyl and aryl is optionally substituted.In certain embodiments, when R¹ is COOR²⁰, R²⁰ is benzyl, methyl, ethyl,or tert-butyl.

In some of the foregoing embodiments of the compounds of formula 23, R¹³is H. In other such embodiments, R¹³ is COOR²⁰ or SO₂R²⁰, wherein R²⁰ isC1-C6 alkyl or aryl-(C1-C6)-alkyl, and each alkyl and aryl is optionallysubstituted. In certain embodiments, when R¹³ is COOR²⁰, R²⁰ is benzyl,methyl, ethyl, or tert-butyl.

In some of the foregoing embodiments of the compounds of formula 23, Xis CH₂. In other such embodiments, X is a bond.

In some of the foregoing embodiments of the compounds of formula 23, Yis O.

In some of the foregoing embodiments, p is 0 or 1 independently at eachoccurrence. In some such embodiments, p is 1.

In some embodiments, the compound of formula 15 is selected from:

and their pharmaceutically acceptable salts.

The pharmaceutically acceptable salts of the compounds of the presentinvention include the conventional nontoxic salts or quaternary ammoniumsalts of the compounds, e.g., from non-toxic organic or inorganic acids.For example, such conventional nontoxic salts include those derived frominorganic acids such as hydrochloride, hydrobromic, sulfuric, sulfamic,phosphoric, nitric, and the like; and the salts prepared from organicacids such as acetic, propionic, succinic, glycolic, stearic, lactic,malic, tartaric, citric, ascorbic, palmitic, maleic, hydroxymaleic,phenylacetic, glutamic, benzoic, salicyclic, sulfanilic,2-acetoxybenzoic, fumaric, toluenesulfonic, methanesulfonic, ethanedisulfonic, oxalic, isothionic, and the like.

In other cases, the compounds of the present invention may contain oneor more acidic functional groups and, thus, are capable of formingpharmaceutically-acceptable salts with pharmaceutically-acceptablebases. The term “pharmaceutically-acceptable salts” in these instancesrefers to the relatively non-toxic, inorganic and organic base additionsalts of compounds of the present invention. These salts can likewise beprepared in situ in the administration vehicle or the dosage formmanufacturing process, or by separately reacting the purified compoundin its free acid form with a suitable base, such as the hydroxide,carbonate or bicarbonate of a pharmaceutically-acceptable metal cation,with ammonia, or with a pharmaceutically-acceptable organic primary,secondary or tertiary amine. Representative alkali or alkaline earthsalts include the lithium, sodium, potassium, calcium, magnesium, andaluminum salts and the like. Representative organic amines useful forthe formation of base addition salts include ethylamine, diethylamine,ethylenediamine, ethanolamine, diethanolamine, piperazine and the like.(See, for example, Berge, et al., supra)

In another aspect, the invention provides a pharmaceutical compositioncomprising at least one compound described in the above embodiments,admixed with at least one pharmaceutically acceptable excipient.

In another aspect, the invention provides a method of treating acondition mediated by the hedgehog pathway, including administering to asubject an effective amount of a compound described herein. Theinvention also provides a method of antagonizing the hedgehog pathway ina subject, including administering to the subject an effective amount ofa compound described herein. The invention also provides a method oftreating cancer in a subject, including administering to a subject atherapeutically effective amount of a compound described herein. Suchcancers include cancers of the central nervous system and cancers of thegastrointestinal tract. The invention further provides a method ofinhibiting activation of a hedgehog pathway in a patient diagnosed witha hyperproliferative disorder, including administering to the patient acompound described herein in an amount sufficient to reduce theactivation of the hedgehog pathway in a cell of the patient.

In another aspect, the invention provides a method to treat a subjectafflicted by excessive activity of a hedgehog pathway, which comprisesadministering to the subject at least one compound described in any ofthe above embodiments, or a pharmaceutically acceptable salt thereof, ora pharmaceutical composition thereof. In some embodiments, the subjectis a subject diagnosed with a hyperproliferative disorder, and in someembodiments, the hyperproliferative disorder is cancer.

Synthesis of Steroidal Alkaloid Compounds

The steroidal alkaloid derivatives described above can be prepareddirectly from naturally occurring steroidal alkaloids or syntheticanalogs thereof. In certain instances, the steroidal alkaloid startingmaterials can be cyclopamine or jervine. These steroidal alkaloids canbe purchased commercially or extracted from Veratrum californicum.

In certain instances, the compounds of the present invention may containa six membered nitrogen containing A-ring (see FIG. 1). These compoundsmay be accessed, as described below, by oxidative cleavage of the A-ringof a steroidal alkaloid with a double bond in the A-ring. Depending onthe position of the double bond in the A-ring the site of cleavage andnitrogen incorporation into the ring may be changed. In the examplesbelow, the double bond is conjugated to a ketone and upon exposure tosodium periodate and potassium permanganate the double is oxidativelycleaved and one carbon is removed from the ring. The resultingketo-ester may be treated with an amine and a reducing agent to form the6-membered lactam. In the example, below ammonium acetate is used toform the 6-membered lactam. The resulting lactam may be furtheralkylated or in the alternative, a primary amine may be used to accesstertiary lactams. The resulting lactams may be reduced to yieldsteroidal alkaloids with an amine in the A-ring.

In certain instances, the compounds of the present invention may containa 7-membered nitrogen containing A-ring. These compounds may be formeddirectly from A-ring oxime-derivatives via the Beckman rearrangement.The rearranged product may be further derivatized by alkylation of thenitrogen of the amide, reduction of the amide to an amine, and the like.In the examples below an A-ring oxime is treated with MsCl and base toaffect the Beckman rearrangement to afford secondary and tertiary A-ringexpanded lactams.

In certain instances, the compounds of the present invention may containa six or seven membered D-ring. Compounds with a 6-membered D-ring areaccessible from certain natural products such as jervine or cyclopamine.Briefly, as illustrated by the example in Scheme A, the seven memberedD-ring analogs may be accessed by cyclopropanating the D-ring of asuitable steroidal alkaloid followed by treating the resultingcyclopropanated product with a Lewis or Brønsted acid to catalyze a ringexpansion rearrangement to yield the seven membered D-ring analogs.

This ring expansion can be performed either before or aftermodifications of the A-ring are accomplished. These ring expandedanalogs may be further functionalized using a variety offunctionalization reactions known in the art. Representative examplesinclude palladium coupling reactions to alkenylhalides or aryl halides,oxidations, reductions, reactions with nucleophiles, reactions withelectrophiles, pericyclic reactions, installation of protecting groups,removal of protecting groups, and the like.

Pharmaceutical Compositions

The compounds disclosed herein or salts thereof may be formulated intocomposition suitable for administration, using one or morepharmaceutically acceptable carriers (additives) and/or diluents. Thepharmaceutical compositions may be specially formulated foradministration in solid or liquid form, including those adapted for thefollowing: (1) oral administration, for example, drenches (aqueous ornon-aqueous solutions or suspensions), tablets, e.g., those targeted forbuccal, sublingual, and systemic absorption, capsules, boluses, powders,granules, pastes for application to the tongue; (2) parenteraladministration, for example, by subcutaneous, intramuscular, intravenousor epidural injection as, for example, a sterile solution or suspension,or sustained-release formulation; (3) topical application, for example,as a cream, ointment, or a controlled-release patch or spray applied tothe skin; (4) intravaginally or intrarectally, for example, as apessary, cream or foam; (5) sublingually; (6) ocularly; (7)transdermally; (8) pulmonarily, or (9) nasally.

Examples of suitable aqueous and nonaqueous carriers which may beemployed in the pharmaceutical compositions include water, ethanol,polyols (such as glycerol, propylene glycol, polyethylene glycol, andthe like), and suitable mixtures thereof, vegetable oils, such as oliveoil, and injectable organic esters, such as ethyl oleate. Properfluidity can be maintained, for example, by the use of coatingmaterials, such as lecithin, by the maintenance of the required particlesize in the case of dispersions, and by the use of surfactants.

These compositions may also contain adjuvants such as preservatives,wetting agents, emulsifying agents, dispersing agents, lubricants,and/or antioxidants. Prevention of the action of microorganisms upon thecompounds disclosed herein may be ensured by the inclusion of variousantibacterial and antifungal agents, for example, paraben,chlorobutanol, phenol sorbic acid, and the like. It may also bedesirable to include isotonic agents, such as sugars, sodium chloride,and the like into the compositions. In addition, prolonged absorption ofthe injectable pharmaceutical form may be brought about by the inclusionof agents which delay absorption such as aluminum monostearate andgelatin.

Methods of preparing these formulations or compositions include the stepof bringing into association a compound with the carrier and,optionally, one or more accessory ingredients. In general, theformulations are prepared by uniformly and intimately bringing intoassociation a compound with liquid carriers, or finely divided solidcarriers, or both, and then, if necessary, shaping the product.

When the compounds disclosed herein are administered as pharmaceuticals,to humans and animals, they can be given per se or as a pharmaceuticalcomposition containing, for example, about 0.1 to 99%, or about 10 to50%, or about 10 to 40%, or about 10 to 30, or about 10 to 20%, or about10 to 15% of active ingredient in combination with a pharmaceuticallyacceptable carrier.

Actual dosage levels of the active ingredients in the pharmaceuticalcompositions may be varied so as to obtain an amount of the activeingredient which is effective to achieve the desired therapeuticresponse for a particular patient, composition, and mode ofadministration, without being toxic to the patient.

The selected dosage level will depend upon a variety of factorsincluding the activity of the particular compound employed, or theester, salt or amide thereof, the route of administration, the time ofadministration, the rate of excretion or metabolism of the particularcompound being employed, the rate and extent of absorption, the durationof the treatment, other drugs, compounds and/or materials used incombination with the particular compound employed, the age, sex, weight,condition, general health and prior medical history of the patient beingtreated, and like factors well known in the medical arts.

In general, a suitable daily dose of a compound disclosed herein will bethat amount of the compound which is the lowest dose effective toproduce a therapeutic effect. Such an effective dose will generallydepend upon the factors described above. Generally, oral, intravenousand subcutaneous doses of the compounds for a patient, when used for theindicated effects, will range from about 0.0001 to about 200 mg, orabout 0.001 to about 100 mg, or about 0.01 to about 100 mg, or about 0.1to about 100 mg per, or about 1 to about 50 mg per kilogram of bodyweight per day.

The compounds can be administered daily, every other day, three times aweek, twice a week, weekly, or bi-weekly. The dosing schedule caninclude a “drug holiday,” i.e., the drug can be administered for twoweeks on, one week off, or three weeks on, one week off, or four weekson, one week off, etc., or continuously, without a drug holiday. Thecompounds can be administered orally, intravenously, intraperitoneally,topically, transdermally, intramuscularly, subcutaneously, intranasally,sublingually, or by any other route. The subject receiving thistreatment is any animal in need, including primates, in particularhumans, and other mammals such as equines, cattle, swine and sheep; andpoultry and pets in general.

Methods of Treatment

Hedgehog signaling is essential in many stages of development,especially in formation of left-right symmetry. Loss or reduction ofhedgehog signaling leads to multiple developmental deficits andmalformations, one of the most striking of which is cyclopia. Manytumors and proliferative conditions have been shown to depend on thehedgehog pathway. The growth of such cells and survival can be affectedby treatment with the compounds disclosed herein. Recently, it has beenreported that activating hedgehog pathway mutations occur in sporadicbasal cell carcinoma (Xie et al. (1998) Nature 391: 90-2) and primitiveneuroectodermal tumors of the central nervous system (Reifenberger etal. (1998) Cancer Res 58: 1798-803). Uncontrolled activation of thehedgehog pathway has also been shown in numerous cancer types such as GItract cancers including pancreatic, esophageal, gastric cancer (Bermanet al. (2003) Nature 425: 846-51, Thayer et al. (2003) Nature 425:851-56) lung cancer (Watkins et al. (2003) Nature 422: 313-317, prostatecancer (Karhadkar et al (2004) Nature 431: 707-12, Sheng et al. (2004)Molecular Cancer 3: 29-42, Fan et al. (2004) Endocrinology 145:3961-70), breast cancer (Kubo et al. (2004) Cancer Research 64: 6071-74,Lewis et al. (2004) Journal of Mammary Gland Biology and Neoplasia 2:165-181) and hepatocellular cancer (Sicklick et al. (2005) ASCOconference, Mohini et al. (2005) AACR conference).

For example, small molecule inhibition of the hedgehog pathway has beenshown to inhibit the growth of basal cell carcinoma (Williams, et al.,2003 PNAS 100: 4616-21), medulloblastoma (Berman et al., 2002 Science297: 1559-61), pancreatic cancer (Berman et al., 2003 Nature 425:846-51), gastrointestinal cancers (Berman et al., 2003 Nature 425:846-51, published PCT application WO 05/013800), esophageal cancer(Berman et al., 2003 Nature 425: 846-51), lung cancer (Watkins et al.,2003. Nature 422: 313-7), and prostate cancer (Karhadkar et al., 2004.Nature 431: 707-12).

In addition, it has been shown that many cancer types have uncontrolledactivation of the hedgehog pathway, for example, breast cancer (Kubo etal., 2004. Cancer Research 64: 6071-4), heptacellular cancer (Patil etal., 2005. 96^(th) Annual AACR conference, abstract #2942 Sicklick etal., 2005. ASCO annual meeting, abstract #9610), hematologicalmalignancies (Watkins and Matsui, unpublished results), basal carcinoma(Bale & Yu, 2001. Human Molec. Genet. 10:757-762 Xie et al., 1998 Nature391: 90-92), medulloblastoma (Pietsch et al., 1997. Cancer Res. 57:2085-88), and gastric cancer (Ma et al., 2005 Carcinogenesis May 19,2005 (Epub)). In addition, investigators have found that small moleculeinhibition of the hedgehog pathway has been shown to ameliorate thesymptoms of psoriasis (Tas, et al., 2004 Dermatology 209: 126-131). Asshown in the Examples, the compounds disclosed herein have been shown tomodulate the hedgehog pathway, and selected compounds have been shown toinhibit tumor growth. It is therefore believed that these compounds canbe useful to treat a variety of hyperproliferative disorders, such asvarious cancers.

Proliferative disorders that can be treated using the methods disclosedherein include: lung cancer (including small cell lung cancer and nonsmall cell lung cancer), other cancers of the pulmonary system,medulloblastoma and other brain cancers, pancreatic cancer, basal cellcarcinoma, breast cancer, prostate cancer and other genitourinarycancers, gastrointestinal stromal tumor (GIST) and other cancers of thegastrointestinal tract, colon cancer, colorectal cancer, ovarian cancer,cancers of the hematopoietic system (including multiple myeloma, acutelymphocytic leukemia, acute myelocytic leukemia, chronic myelocyticleukemia, chronic lymphocytic leukemia, Hodgkin lymphoma, andnon-Hodgkin lymphoma, and myelodysplastic syndrome), polycythemia Vera,Waldenstrom's macroglobulinemia, heavy chain disease, soft-tissuesarcomas, such as fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, squamous cell carcinoma, basal cell carcinoma,melanoma, and other skin cancers, adenocarcinoma, sweat gland carcinoma,sebaceous gland carcinoma, papillary carcinoma, papillaryadenocarcinomas, stadenocarcinoma, medullary carcinoma, bronchogeniccarcinoma, renal cell carcinoma, hepatoma, bile duct carcinoma,choriocarcinoma, seminoma, embryonal carcinoma, Wilms' tumor, cervicalcancer, uterine cancer, testicular cancer, bladder carcinoma, and othergenitourinary cances, epithelial carcinoma, glioma, astrocytoma,medulloblastoma, craniopharyngioma, ependymoma, pinealoma,hemangioblastoma, acoustic neuroma, oligodendroglioma, meningioma,neuroblastoma, retinoblastoma, endometrial cancer, follicular lymphoma,diffuse large B-cell lymphoma, mantle cell lymphoma, hepatocellularcarcinoma, thyroid cancer, gastric cancer, esophageal cancer, head andneck cancer, small cell cancers, essential thrombocythemia, agnogenicmyeloid metaplasia, hypereosinophilic syndrome, systemic mastocytosis,familiar hypereosinophilia, chronic eosinophilic leukemia, thyroidcancer, neuroendocrine cancers, and carcinoid tumors. Additionaldisorders include Gorlin's syndrome and psoriasis The subject receivingthis treatment is any animal in need, including primates, in particularhumans, and other mammals such as equines, cattle, swine and sheep; andpoultry and pets in general.

The hedgehog inhibitors disclosed herein can be combined with othercancer treatments. For example, they can be combined with surgicaltreatments; radiation; biotherapeutics (such as interferons,cytokines—e.g. Interferon α, Interferon γ, and tumor necrosis factor,hematopoietic growth factors, monoclonal serotherapy, vaccines andimmunostimulants); antibodies (e.g. Avastin, Erbitux, Rituxan, andBexxar); endocrine therapy (including peptide hormones, corticosteroids,estrogens, androgens and aromatase inhibitors); anti-estrogens (e.g.Tamoxifen, Raloxifene, and Megestrol); LHRH agonists (e.g. goscrclin andLeuprolide acetate); anti-androgens (e.g. flutamide and Bicalutamide);gene therapy; bone marrow transplantation; photodynamic therapies (e.g.vertoporfin (BPD-MA), Phthalocyanine, photosensitizer Pc4, andDemethoxy-hypocrellin A (2BA-2-DMHA)); and chemotherapeutics.

Examples of chemotherapeutics include gemcitabine, methotrexate, taxol,mercaptopurine, thioguanine, hydroxyurea, cytarabine, cyclophosphamide,ifosfamide, nitrosoureas, cisplatin, carboplatin, mitomycin,dacarbazine, procarbizine, etoposides, prednisolone, dexamethasone,cytarbine, campathecins, bleomycin, doxorubicin, idarubicin,daunorubicin, dactinomycin, plicamycin, mitoxantrone, asparaginase,vinblastine, vincristine, and vinorelbine. Additional agents includenitrogen mustards (e.g. cyclophosphamide, Ifosfamide, Trofosfamide,Chlorambucil, Estramustine, and Melphalan), nitrosoureas (e.g.carmustine (BCNU) and Lomustine (CCNU)), alkylsulphonates (e.g. busulfanand Treosulfan), triazenes (e.g. Dacarbazine and Temozolomide), platinumcontaining compounds (e.g. Cisplatin, Carboplatin, and oxaliplatin),vinca alkaloids (e.g. vincristine, Vinblastine, Vindesine, andVinorelbine), taxoids (e.g. paclitaxel and Docetaxol), epipodophyllins(e.g. etoposide, Teniposide, Topotecan, 9-Aminocamptothecin,Camptoirinotecan, Crisnatol, Mytomycin C, and Mytomycin C),anti-metabolites, DHFR inhibitors (e.g. methotrexate and Trimetrexate),IMP dehydrogenase Inhibitors (e.g. mycophenolic acid, Tiazofurin,Ribavirin, and EICAR), ribonucleotide reductase Inhibitors (e.g.hydroxyurea and Deferoxamine), uracil analogs (e.g. Fluorouracil,Floxuridine, Doxifluridine, Ratitrexed, and Capecitabine), cytosineanalogs (e.g. cytarabine (ara C), Cytosine arabinoside, andFludarabine), purine analogs (e.g. mercaptopurine and Thioguanine),Vitamin D3 analogs (e.g. EB 1089, CB 1093, and KH 1060), isoprenylationinhibitors (e.g. Lovastatin), dopaminergic neurotoxins (e.g.1-methyl-4-phenylpyridinium ion), cell cycle inhibitors (e.g.staurosporine), actinomycins (e.g. Actinomycin D and Dactinomycin),bleomycins (e.g. bleomycin A2, Bleomycin B2, and Peplomycin),anthracyclines (e.g. daunorubicin, Doxorubicin (adriamycin), Idarubicin,Epirubicin, Pirarubicin, Zorubicin, and Mitoxantrone), MDR inhibitors(e.g. verapamil), Ca²⁺ ATPase inhibitors (e.g. thapsigargin), imatinib,thalidomide, lenalidomide, erlotinib, gefitinib, sorafenib, andsunitinib, and proteasome inhibitors, including bortezomib.

When the hedgehog inhibitors disclosed herein are administered incombination with other treatments, such as additional therapeutics orwith radiation or surgery, the doses of each agent or therapy will inmost instances be lower than the corresponding dose for single-agenttherapy. Also, in general, the hedgehog inhibitors described herein andthe second therapeutic agent do not have to be administered in the samepharmaceutical composition, and may, because of different physical andchemical characteristics, be administered by different routes. Forexample, one compound can be administered orally, while the secondtherapeutic is administered intravenously. The determination of the modeof administration and the advisability of administration, wherepossible, in the same pharmaceutical composition, is well within theknowledge of the skilled clinician. The initial administration can bemade according to established protocols known in the art, and then,based upon the observed effects, the dosage, modes of administration andtimes of administration can be modified by the skilled clinician.

The hedgehog inhibitor and the second therapeutic agent and/or radiationmay be administered concurrently (e.g., simultaneously, essentiallysimultaneously or within the same treatment protocol) or sequentially(i.e., one followed by the other, with an optional time interval inbetween), depending upon the nature of the proliferative disease, thecondition of the patient, and the actual choice of second therapeuticagent and/or radiation to be administered.

If the hedgehog inhibitor, and the second therapeutic agent and/orradiation are not administered simultaneously or essentiallysimultaneously, then the optimum order of administration may bedifferent for different conditions. Thus, in certain situations thehedgehog inhibitor may be administered first followed by theadministration of the second therapeutic agent and/or radiation; and inother situations the second therapeutic agent and/or radiation may beadministered first followed by the administration of a hedgehoginhibitor. This alternate administration may be repeated during a singletreatment protocol. The determination of the order of administration,and the number of repetitions of administration of each therapeuticagent during a treatment protocol, is well within the knowledge of theskilled physician after evaluation of the disease being treated and thecondition of the patient. For example, the second therapeutic agentand/or radiation may be administered first, especially if it is acytotoxic agent, and then the treatment continued with theadministration of a hedgehog inhibitor followed, where determinedadvantageous, by the administration of the second therapeutic agentand/or radiation, and so on until the treatment protocol is complete.

EXEMPLIFICATION

The invention now being generally described, it will be more readilyunderstood by reference to the following examples, which are includedmerely for purposes of illustration of certain aspects and embodimentsof the present invention, and are not intended to limit the invention.

Example 1

Step A

Cyclopamine 2 (5.02 g, 12.2 mmol, 1.0 eq) was dissolved in anhydrouspyridine (25 mL) DMAP (300 mg, 2.44 mmol, 0.2 eq.) and triethyl amine(5.5 mL, 39.1 mmol, 3.2 eq) were added, followed by BtO-Cbz (10.5 g,39.1 mmol, 3.2 eq) and the mixture was heated at 40° C. for 2 h. Themixture was cooled to rt, treated with 30 mL water, heated to get ahomogeneous solution and allowed to cool to rt. The white, precipitatethat formed was collected by filtration, the filter cake was washed withportions of water (3×50 mL), and dried in air to afford 9.53 g of crudematerial which crystallized from toluene/heptanes (1:9, 70 mL) to give6.75 g of the desired product.

Step B

Bis(2,6-dimethylphenyl)phosphate (6.76 g, 22.1 mmol, 3 eq) was dissolvedat rt with anhydrous DCM (50 mL) and azeotroped (2×). The resultingsolid was placed under high vacuum for 12 h. The solid was suspended inDCM (50 mL) to yield a clear solution (Flask A). Bis-Cbz protectedcyclopamine (5 g, 7.35 mmol, 1 eq) was dissolved in anhydrous DCM (50mL) and azetroped (2×). The resulting white foam was placed under highvacuum for 12 h. The dried Bis-Cbz protected cyclopamine was dissolvedin anhydrous DCM (15 mL) (Flask B). In a glove box, a flame dried 500 mLflask was charged with diethylzinc (2.63 g, 21.3 mmol, 2.9 eq). Theflask was sealed with a septum and taken out of the dry box. The flaskwas placed under a balloon of Ar and charged with anhydrous DCM (50 mL)(Flask C). Flask B was added via cannula to flask C over 15 min. Thereaction was allowed to stir at rt for 20 min A clear solution wasobtained. Flask A was transferred to the reaction flask C via cannulaover 10 min. The reaction was stirred for an additional 5 min resultingin a slightly hazy yellow solution. Diiodomethane (1.78 mL, 22.1 mmol, 3eq) was added at rt over 1 min. The reaction was allowed to stir for 24h. The reaction was quenched by the addition of a saturated aqueousNH₄Cl solution. The layers were separated and the aqueous layers wereback extracted with DCM. The combined organic layers were washed withsaturated aqueous NH₄Cl solution (1×), 5% NaHCO₃ (2×), 10% Na₂SO₃ (1×).The organic solvent was dried over Na₂SO₄, filtered, and evaporated todryness to give a foamy solid. Purification by flash silica gelchromatography (hexanes/EtOAc 95:5 to 8:2) yielded 3.8 g of the desiredmaterial.

Step C

A round-bottom flask was charged with Bis-Cbz-protectedcyclopropylcyclopamine (2 g, 2.88 mmol, 1 eq), MeOH (15 mL), and THF (5mL) and vigorously stirred with 2N NaOH (2 mL) at 55° C. for 3 h. TheTHF and MeOH were removed under reduced pressure and the residue wasextracted with EtOAc (3×). The combined organic layers were washed withbrine, dried over Na₂SO₄, filtered, and evaporated to dryness to give afoamy solid. Purification by flash silica gel chromatography(hexanes/EtOAc 9:1 to 8:2) to afford 1.1 g of the desired material.

Step D

A round-bottom flask was charged with N-Cbz-cyclopropylcyclopamine (2.3g, 4.1 mmol, 1 eq), Al(OBu)₃ (1.4 g, 5.76 mmol, 1.4 eq), toluene (30mL), and 2-butanone (30 mL). The mixture was heated at 75° C. under Arfor 10 h. The reaction was quenched by the addition of an aqueous 20%Rochelle's salt solution. The biphasic mixture was stirred at 40° C. for20 min. The layers were separated and the aqueous layer was extractedthree times with EtOAc/toluene (1:1). The combined organic layers werewashed with 20% Rochelle's salt, dried over Na₂SO₄, filtered, andconcentrated to dryness by two successive azeotropic distillations withheptanes. The crude material was purified using silica gel flashchromatography (hexanes/EtOAc 4:1) to afford 859 mg of the desiredproduct.

Step E

The enone derivative (350 mg, 0.63 mmol, 1 eq) was dissolved in t-BuOH(5 mL) then a solution of Na₂CO₃ (100 mg, 0.94 mmol, 1.5 eq) in water (5mL) was added. The mixture was heated to 80° C. and charged with asolution of NaIO₄ (0.94 g, 4.4 mmol, 7 eq) and KMnO₄ (7 mg, 0.043 mmol,0.07 eq) in water (5 mL). After 60 min, the mixture was cooled to rt.The basic mixture was acidified with 2N HCl, and extracted with EtOAc(3×). The combined organic layers were washed with saturated aqueousNH₄Cl, dried over Na₂SO₄, filtered and concentrated to dryness. Thematerial was chased a couple times with MTBE. The material (373 mg) wasdissolved in toluene/MeOH (10 mL; 4:1, HPLC grade) and treated with of2M TMSCHN₂ in hexanes (630 μL, 1.2 mmol, 2 eq). Bubbling was observedand the bright yellow color persisted. Nitrogen was bubbled through thesolution then the solution was concentrated to dryness. The crudematerial (366 mg) was purified using silica gel flash chromatography(hexanes/EtOAc 95:5 to 4:1) to afford the desired material as a whitefoam (245 mg).

Step F

The compound 7 (237 mg, 0.4 mmol, 1 eq) was dissolved in MeOH (5 mL) andtreated with NH₄OAc (1.2 g, 16 mmol, 40 eq) and sodium cyanoborohydride(251 mg, 4 mmol, 10 eq) at rt for 3 h. Then, NH₄OAc (600 mg, 8 mmol, 20eq) was added to the reaction mixture, which was warmed to 50° C. andstirred for 4 h. Saturated aqueous NaHCO₃ was added and MeOH wasdistilled under reduced pressure. The residue was extracted with EtOAc(3×). The combined organic layers were washed with 1N NaOH (1×), thensaturated NH₄Cl (1×), then the layers clearly separated. The organiclayer was dried over Na₂SO₄, filtered, and concentrated to dryness. Thecrude material (257 mg) was purified using flash silica gel flashchromatography (hexanes/EtOAc 1:9 to 100% EtOAc) to give 5β-reducedlactam (82 mg compound 8) followed by 5α-reduced lactam (89 mg, compound9).

Step G

A cooled (−78° C.) solution of compound 9 (82 mg, 0.15 mmol, 1 eq) inanhydrous DCM (3 mL) was treated with BF₃-OEt₂ (55 μL, 0.45 mmol, 3 eq).The mixture was stirred at −78° C. for 15 min then warmed up to 0° C.and stirred for 30 min. The reaction was quenched by the addition ofsaturated aqueous NaHCO₃. The residue was extracted with DCM (3×). Thecombined DCM layers were washed with saturated aqueous NaHCO₃, water,dried over Na₂SO₄, filtered, and evaporated to dryness. The crudematerial (71 mg) was purified by silica gel flash chromatography(hexanes/EtOAc 1:9 to 100% EtOAc) to afford the desired 5α-reducedlactam product as solidifying oil (48 mg).

Step H

Compound 10 (42 mg, 0.075 mmol, 1 equiv.) was dissolved in EtOAc (3 mL)and treated with Pd/C 10% (8 mg, wet, Aldrich Degussa type E101 lot08331KC). The flask was sealed and purged with hydrogen (3×) and stirredfor 10 h under 1 atm of hydrogen. The mixture was filtered through a 0.2micron AcrosDisc filter to give 46 mg of crude material. Purification bysilica gel flash chromatography (DCM to DCM/MeOH 92:8) afforded 20 mg ofpure material ([M+H]=427.4 m/z).

Example 2

Compound 11 was synthesized according to the procedure described inexample 1, using compound 8 in place of compound 9 in steps G and H.

Example 3

Step A

To a solution of diethyl zinc (572 mg, 482 μL, 4.63 mmol, 3 eq) in DCM(5.0 mL) at −20° C. was added a solution ofbis-(2,6-Dimethylphenyl)phosphoric acid (1.42 g, 4.63 mmol, 3 eq) in DCM(15 mL) while maintaining the reaction temperature below −8° C. Thesolution was aged for 15 min. at 0° C., neat diiodomethane (1.24 g, 374μL, 3 eq) was added, and the mixture was aged for 15 min. at 0° C.before adding a solution of (Bis-CBz-cyclopamine, 1.05 g, 1.54 mmol, 1.0eq), in DCM (10 mL). The cooling bath was replaced by a water bath at rtand maintained at rt for 4.5 h. The mixture was cooled to −76° C. with adry ice-acetone bath and treated drop wise with methanesulfonic acid DCMsolution (0.6 mL 50% v/v solution 4.63 mmol, 3.0 eq) while maintainingthe reaction temperature below −74° C. The mixture was aged for 15-20min. and quenched drop wise with morpholine (2.69 g, 2.70 mL, 20 eq)maintaining the reaction temperature below −65° C. The cooling bath wasremoved, the reaction mixture was stirred for 16-18 h., the whiteprecipitate was filtered off, and the filtrate was successively washedwith 2.0 m HCl (2×20 mL), satd. sodium bicarbonate (2×20 mL), water(2×20 mL) and brine (20 mL) It was then dried over magnesium sulfate,concentrated in vacuo to dryness and the crude was purified by silicagel flash chromatography (hexanes/EtOAc 17:3→4:1) to afford 924 mg (1.33mmol, 86%) of the desired product.

Step B

To a solution of compound 13 (4.05 g, 5.83 mmol, 1 eq) in a solution ofEtOAc:toluene (2:1, 60 mL) was added 20% palladium hydroxide on carbon(823 mg, 0.583 mmol, 0.1 eq). The flask was evacuated and filled withhydrogen three times. The mixture was stirred under an atmosphere ofhydrogen for 1 h. Neat ethylene diamine (0.38 mL) was added, and themixture was stirred for 1 h., before the catalyst was filtered off. Thefilter cake was washed twice with EtOAc:toluene (2:1, 12 mL). Thecombined filtrates were washed with a 2% aqueous solution of ethylenediamine (3×20 mL), dried over sodium sulfate and concentrated in vacuoto give 2.46 g as a white crystalline solid.

Step C

A round bottom flask was sequentially charged with the homo-allylicalcohol 14 (7.50 g, 17.6 mmol, 1 eq), aluminum tert-butoxide (6.10 g,24.8 mmol, 1.4 eq), anhydrous toluene (115 mL), and 2-butanone (90 g,1.24 mol, 7 eq). The suspension was heated under a nitrogen atmosphereto 75° C. for 16 h. The reaction temperature was then allowed to cool to49° C. Aqueous 20% (w/w) potassium sodium tartrate solution (226 g) wasadded to the stirred suspension. The suspension was stirred at rt for3.5 h. The layers were separated. The organic layer was washed withaqueous 20% Rochelle's salt (2×250 mL) and water (225 mL), then driedover sodium sulfate and filtered. The residue was rinsed with toluene(30 mL) and discarded. The combined organics were concentrated todryness. Residual reaction solvents were removed from the material byconcentrating from 2-propanol (250 mL added portion-wise) to a finalsolution mass of 44 g. Solvent exchange from 2-propanol to n-heptane(275 mL added portion-wise) to a final solution mass of 41 g fullyprecipitated the desired product. The suspension was diluted withadditional n-heptane (40 mL), stirred at rt for 1 h, and filtered. Theproduct was washed with n-heptane (17 mL) and dried to afford 5.4 g ofthe desired product.

Step D

A round-bottom flask was charged with starting material (110 mg, 0.26mmol, 1 eq) and 10% palladium on carbon (106 mg). The solids weresuspended in pyridine (4 mL). The suspension was placed under hydrogenatmosphere (1 atm) and the mixture was stirred overnight at rt. Thereaction mixture was filtered through celite and the filtrateconcentrated in vacuo. The crude material was purified using silica gelflash chromatography (MeOH/DCM 5:95) to afford 93 mg of the desiredcompound.

Step E

A round-bottom flask was charged with compound 16 (4.23 g, 9.94 mmol, 1eq) and THF (60 mL). Triethylamine (6.92 mL, 49.7 mmol, 5.0 eq) andbenzyl chloroformate (1.54 mL, 10.93 mmol, 1.1 eq) were added and themixture was stirred for 1 h at rt. The reaction mixture was partitionedbetween saturated aqueous bicarbonate (100 mL) and EtOAc (100 mL). Thephases were separated and the organics were dried (Na₂SO₄) andconcentrated to dryness. The crude material was purified using silicagel flash chromatography (EtOAc/Hexanes 2:98→14:86) to give 3.75 g ofmaterial.

Step F

An ethanol solution (5 ml) of compound 17 (185 mg, 0.3 mmol, 1 eq) wastreated with hydroxylamine hydrochloride (140 mg, 2 mmol, 6 eq), sodiumacetate (160 mg, 2 mmol, 6 eq), and water (0.5 ml), and the mixture wasstirred at rt for 1 hr. The mixture was split between EtOAc and water(50 ml each). The organic layer was washed with brine (30 ml), driedover sodium sulfate, and concentrated to a white residue. The crudeproduct was purified by silica gel chromatography (ether/hexanes2:3→1:1) to give 193 mg of oxime 18.

Step G

Compound 18 (50 mg, 0.087 mmol, 1.0 eq) was dissolved in dry pyridine(1.0 mL) and at 0° C. treated with methanesulfonyl chloride (20.0 mg,0.174 mmol, 2.0 eq). After stirring for 2 h, the solution was warmed tort and treated with 5N sodium hydroxide (0.3 ml, 1.5 mmol, 18 eq) andstirred for 1 h. The mixture was split between EtOAc (30 mL) and 1Maqueous hydrogen chloride (15 mL). The organic layer was washed withwater, washed with brine, dried over sodium sulfate, and concentrated toa clear oil. The mixture of lactams was purified by silica gelchromatography (80-100% ethyl acetate/hexanes, then 1% methanol in ethylacetate) to afford a mixture of the lactam regioisomers as a clear oil(34 mg, 68% yield).

The product carbamate lactams were dissolved in EtOAc (7 ml) in a flaskwith stir bar and rubber septum. The solution was sparged with nitrogen,and 10% Pd/C (wet, Degussa type E101, Aldrich, 25 mg) was added. Thismixture was sparged with nitrogen and then hydrogen gas and stirred atrt for 3 h. The mixture was then sparged with nitrogen, filtered througha 0.45 μM polyethylene membrane and concentrated to a clear oil. The oilwas purified by silica gel flash chromatography (0.5% ammoniumhydroxide/2-20% MeOH/DCM), and the pure fractions were concentrated togive an oil that was lyophilized from 7% water/tbutanol, to afford a 1:1mixture of unseparated lactams, as a white powder (19 mg: [M+H]=441.6m/z).

Example 4

Compound 19 was made using procedures similar to those described inExample 3.

Example 5

Step A

Compound 15 (2.0 g, 4.7 mmol, 1.0 eq) was dissolved in DCM (20.0 mL) andTHF (20.0 mL) and at rt treated with triethylamine (3.3 ml, 23.6 mmol,5.0 eq) and Cbz-Cl (0.73 ml, 5.2 mmol, 1.1 eq). 4-Dimethylaminopyridine(50 mg) was added, and the mixture was stirred for 60 min at rt. Themixture was split between EtOAc (200 mL) and 5% aqueous sodiumbicarbonate (150 mL). The organic layer was washed with brine (50 mL),dried over sodium sulfate, and concentrated. Purification of the residueby silica gel flash chromatography (5→40% EtOAc/hexanes) afforded thedesired carbamate as a white foam. (2.4 g).

Step B

Compound 20 (200.0 mg, 0.36 mmol, 1.0 eq) was dissolved in EtOH (3.0 mL)and water (0.3 mL) and at rt treated with hydroxylamine hydrochloride(150 mg, 2.1 mmol, 6.0 eq) and sodium acetate (176 mg, 2.1 mmol, 6 eq).The mixture was heated at 70° C. for 10 min. The mixture was splitbetween EtOAc (30 mL) and water (15 mL). The organic layer was washedwith brine (15 mL), dried over sodium sulfate, and concentrated to givethe oxime 21 as a white foam. (202 mg).

Step C

Compound 21 (200.0 mg, 0.34 mmol, 1.0 eq) was dissolved in dry pyridine(1.0 mL) and at 0° C. treated with methanesulfonyl chloride (120.0 mg,1.05 mmol, 3.0 eq). After stirring for 2 h, the solution was warmed tort and treated with 5 N sodium hydroxide (0.75 ml, 4.25 mmol, 12 eq) andstirred for 12 h. The mixture was split between EtOAc (30 mL) and 1 maqueous HCl (15 mL). The organic layer was washed with water and thenbrine (15 mL each), dried over sodium sulfate, and concentrated to givethe oxime O-methanesulfonate as a clear oil. This oil was suspended inMeOH (5 mL), treated with concentrated aqueous HCl (0.75 mL), and heatedat 60° C. for 2 h. This dark brown mixture was concentrated in vacuo andpurified by silica gel flash chromatography (50→100% EtOAc/hexanesfollowed by 1→5% MeOH in EtOAc) to give the unsaturated lactam as anisomerically pure white solid (45 mg).

The product carbamate lactam was dissolved in pyridine (7 mL) in a flaskwith stir bar and rubber septum. The solution was sparged with nitrogen,and 10% Pd/C (wet, Degussa type E101, Aldrich, 25 mg) was added. Thismixture was sparged with nitrogen and then hydrogen gas and stirred atrt for 48 h. The mixture was then sparged with nitrogen, filteredthrough a 0.45 μm polyethylene membrane and concentrated to a clear oil.The oil was purified by silica gel chromatography (0.5% ammoniumhydroxide/2→20% MeOH/DCM), and the pure fractions were concentrated togive an oil that was lyophilized from 7% water/t-butanol, affording theproduct as a white powder (14 mg: [M+H]=441.7 m/z).

Example 6

Step A

A dry round-bottom flask was charged with KOtBu (0.57 g, 5.1 mmol, 7 eq)and tBuOH (6 mL) and the solution was stirred at rt for 10 min. Compound17 (0.3 g, 0.73 mmol, 1 eq) was added and stirred for 5 min. The whitesuspension became a yellow clear solution. Ethyl formate (0.35 mL, 4.4mmol, 6 eq) was added dropwise, and the solution became slightly opaqueand produced bubbles. The slurry was stirred at rt for 48 h. The mixturewas then portioned between MTBE/1% NaOH (2×20 mL). The aqueous layer wasacidified with 2 N HCl until the pH reached 5, then extracted withchloroform (2×). The combined organic layers were washed with water,dried over Na₂SO₄, and concentrated to dryness to give 200 mg paleyellow foam. This material was used without further purification in thenext step.

Step B

A round-bottom flask was charged with compound 22 (2.0 g, 3.40 mmol, 1eq) and was dissolved in toluene (25 mL). The reaction was charged withDDQ (0.849 g, 3.74 mmol, 1.1 eq). The mixture was stirred for 0.5 hr atrt. The reaction mixture was then concentrated in vacuo to 10% theoriginal volume. The residue was purified using silica gel flashchromatography (EtOAc/hexanes 10% 20%) to afford the desired material.

Step C

A round-bottom flask was charged with compound 23 (1.10 g, 1.88 mmol, 1eq) and was dissolved in toluene (25 mL). The reaction was charged withWilkinson's catalyst (1.77 g, 1.92 mmol, 1.02 eq). The mixture washeated to 80° C. stirred for 0.5 h. The reaction mixture was cooled andconcentrated in vacuo to 10% the original volume. The residue waspurified using silica gel flash chromatography (EtOAc/hexanes 10%→15%)to afford the desired material.

Step D

A round-bottom flask was charged with compound 24 (750 mg, 1.34 mmol, 1eq) and was dissolved in EtOH (10 mL) and water (1 mL). The reaction wascharged with hydroxylamine hydrochloride (662 mg, 8.07 mmol, 6.0 eq) andsodium acetate (561 mg, 8.07 mmol, 6 eq). The mixture was stirred for0.5 h at rt. The reaction mixture was partitioned between water andEtOAc. The organic was separated, dried and concentrated to dryness. Theresidue was purified using silica gel flash chromatography(EtOAc/hexanes 10%→25%) to afford the desired material.

Step E

A round-bottom flask was charged with compound 25 (770 mg, 1.34 mmol, 1eq) and was dissolved in pyridine (10 mL). The reaction was charged withmethanesulfonylchloride (462 mg, 4.03 mmol, 3.0 eq). The mixture wasstirred for 0.5 h at rt. The reaction mixture was partitioned between anaqueous solution of 1N HCl and EtOAc. The organic was separated, driedand concentrated to dryness. The residue was purified using silica gelflash chromatography (EtOAc/hexanes 15%→20%) to afford the desiredmaterial.

Step F

A round-bottom flask was charged with compound 26 (765 mg, 1.18 mmol, 1eq) and was dissolved in MeOH (30 mL). The reaction was charged withconcentrated HCl (300 mg, 3.54 mmol, 7.0 eq). The mixture was heated to60° C. and stirred for 17 h. The reaction mixture was partitionedbetween a saturated aqueous solution of sodium bicarbonate and EtOAc.The organic was separated, dried and concentrated to dryness. Theresidue was purified using silica gel flash chromatography(EtOAc/hexanes 50% 90%) to afford the desired material.

Step G

A round-bottom flask was charged with compound 27 (142 mg, 0.248 mmol, 1eq) and 10% palladium on carbon (30 mg). The solids were suspended inEtOH (3 mL). The suspension was placed under hydrogen atmosphere and themixture was stirred for 2 h at rt. The reaction mixture was filtered oncelite and the filtrate concentrated to dryness. The residue waspurified using silica gel flash chromatography (MeOH/DCM 0%→5%) toafford the desired material. ([M+H]=441.5 m/z).

Example 7

Step A

A round-bottom flask was charged with starting material (51 mg, 0.09mmol, 1 eq). The solid was dissolved in 5 mL of tetrahydrofuran. Thesolution was cooled to −78° C. A 0.5M solution of potassiumbis(trimethylsilyl)amide in toluene (0.207 mL, 0.104 mmol, 1.2 eq.) wascharged and the solution was stirred for 0.5 hr at −78° C. The reactionwas then charged with methyliodide (11 uL, 0.179 mmol, 2 eq.) and thereaction was warmed to 25° C. The reaction was stirred o/n then chargedwith water and ethyl acetate. The organic was separated, dried andconcentrated to dryness. The residue was dissolved in DCM and was loadedonto a SiO₂. Elution with 50 to 90% EtOAc/hexanes gave Compound 32([M+H]=587.8 m/z).

Step B

A round-bottom flask was charged with starting material (50 mg, 0.085mmol, 1 eq) and 10% palladium on carbon (10 mg). The solids weresuspended in 3 mL of ethanol. The suspension was placed under hydrogenatmosphere and the mixture was stirred for 4 hrs at 25° C. LCMS showcomplete disappearance of starting material. The reaction mixture wasfiltered on celite and the filtrate concentrated to dryness. The residuewas dissolved in DCM and was loaded onto a SiO₂. Elution with 0 to 8%MeOH/DCM gave the desired product Compound 31 ([M+H]=455.5 m/z).

Example 8

A round-bottom flask was charged with Compound 31 (10 mg, 0.022 mmol, 1eq) and dichloromethane (1 mL). The solution was charged withtriethylamine (3.2 uL, 0.066 mmol, 3 eq.) and methane sulfonylchloride(14 uL, 0.088 mmol, 4 eq.) were charged and the solution was stirred for0.5 hr. The reaction mixture was partitioned between a solution asaturated aqueous solution of sodium bicarbonate and ethyl acetate. Theorganic was separated, dried and concentrated to dryness. The residuewas dissolved in DCM and was loaded onto a SiO₂. Elution with 75 to 90%EtOAc/hexanes gave the desired material Compound 35 ([M+H]=533.8 m/z).

Example 9

A round-bottom flask was charged with Compound 12 (16 mg, 0.036 mmol, 1eq) and dichloromethane (1 mL). The solution was charged withtriethylamine (8.0 uL, 0.109 mmol, 3 eq.) and methane sulfonylchloride(20 uL, 0.149 mmol, 4 eq.) were charged and the solution was stirred for0.5 hr. The reaction mixture was partitioned between a solution asaturated aqueous solution of sodium bicarbonate and ethyl acetate. Theorganic was separated, dried and concentrated to dryness. The residuewas dissolved in DCM and was loaded onto a SiO₂. Elution with 75 to 90%EtOAc/hexanes gave the desired material Compound 40 ([M+H]=519.8 m/z).

Example 10

A round-bottom flask was charged with starting material (1.15 g, 2.06mmol, 1 eq) and ethanol (15 mL). The solution was charged with sodiumacetate (1.015 g, 12.37 mmol, 6 eq.) and N-methylhydroxylamine HCl(0.189 g, 2.27 mmol, 1.1 eq.) were charged and the solution was heatedto 70° C. and stirred for 6 hrs. The reaction mixture was cooled to rtand concentrated. The residue was partitioned between water and ethylacetate. The organic was separated, dried and concentrated to dryness.The residue was dissolved in DCM and was loaded onto a SiO₂. Elutionwith 0 to 10% MeOH/DCM gave the desired material ([M+H]=587.9 m/z).

A round-bottom flask was charged with starting material (1.10 g, 1.875mmol, 1 eq) and dissolved in pyridine (15 mL) and water (0.7 mL). Thesolution was charged with tosyl chloride (0.430 g, 2.249 mmol, 1.2 eq.)was charged and the solution the solution was stirred at rt for 0.5 hr.The reaction mixture was cooled to rt and concentrated. The residue waspartitioned between water and MTBE. The organic was separated, dried andconcentrated to dryness. The residue was dissolved in DCM and was loadedonto a SiO₂. Elution with 10 to 80% EtOAc/Hex gave the desired material([M+H]=587.9 m/z).

A round-bottom flask was charged with starting material (100 mg, 0.170mmol, 1 eq) and 10% palladium on carbon (50 mg). The solids weresuspended in 3 mL of ethyl acetate. The suspension was placed underhydrogen atmosphere and the mixture was stirred for 4 hrs at 25° C. Thereaction mixture was filtered on celite and the filtrate concentrated todryness. The residue was dissolved in DCM and was loaded onto a SiO₂.Elution with 0 to 10% MeOH/DCM gave the desired Compound 41 ([M+H]=453.5m/z).

Example 11

Step A

A dried flask was charged with 46 (355 mg, 0.806 mmol, 1 equiv.) and dryTHF (5 mL) and pyridine (326 uL, 4.03 mmol, 5 equiv.). The cooled (0°C.) solution was treated with benzoyl peroxide (585 mg, 2.42 mmol, 3equiv.). The mixture was stirred for 1 h at 0° C., then the solution wasgradually warmed to 25° C. After 2 h, the mixture was diluted with EtOAcand washed with aqueous saturated NaHCO₃ solution. The aqueous layer wasextracted once more with EtOAc. The combined organic layer were driedover Na₂SO₄, filtered, and evaporated to dryness. The oil was dissolvedin CH₂Cl₂ and purified by SiO₂ column eluting with hexane/EtOAc (40 to100%) to give 238 mg of desired compound 47.

Step B

A round-bottom flask was charged with 47 (229 mg, 0.41 mmol, 1 equiv.)and MeOH (5 mL). The solution was treated at 25° C. in presence of 2 NKOH (1 mL, 2 mmol, 5 equiv.). The mixture was stirred for 3 h. Thesolvent was removed by nitrogen stream and the solution was neutralizedwith 500 uL of 1N HCl. The aqueous layer was extracted with threeportions of CH₂Cl₂. Combined organic layers were dried over Na₂SO₄,filtered, and concentrated to dryness. The crude material (220 mg) wasdissolved with CH₂Cl₂, loaded onto a SiO₂ column (12 g) and eluted withCH₂Cl₂/MeOH (0% to 100%) to give the hydroxylamine 45. The materialrecrystallized from heptane/2-propanol to give desired material 3([M+H]=547.5 m/z).

Compound 50 was prepared using techniques similar to those describedabove.

Example 12 Inhibition of the Hedgehog Pathway in Cell Culture UsingAnalogs of Cyclopamine

Hedgehog pathway specific cancer cell killing effects may be ascertainedusing the following assay. C3H10T1/2 cells differentiate intoosteoblasts when contacted with the sonic hedgehog peptide (Shh-N). Upondifferentiation; these osteoblasts produce high levels of alkalinephosphatase (AP) which can be measured in an enzymatic assay (Nakamura,et al., BBRC (1997) 237:465). Compounds that block the differentiationof C3H10T1/2 into osteoblasts (a Shh dependent event) can therefore beidentified by a reduction in AP production (van der Horst, et al., Bone(2003) 33:899). The assay details are described below. The results (EC₅₀for inhibition) of the differentiation assay are shown below in Table 1.

Assay Protocol

Cell Culture

Mouse embryonic mesoderm fibroblasts C3H10T1/2 cells (obtained fromATCC) were cultured in Basal MEM Media (Gibco/Invitrogen) supplementedwith 10% heat inactivated FBS (Hyclone), 50 units/ml penicillin and 50ug/ml streptomycin (Gibco/Invitrogen) at 37° C. with 5% CO₂ in airatmosphere.

Alkaline Phosphatase Assay

C3H10T1/2 cells were plated in 96 wells with a density of 8×10³cells/well. Cells were grown to confluence (72 hrs). After sonicHedgehog (250 ng/ml), and/or compound treatment, the cells were lysed in110 μL, of lysis buffer (50 mM Tris pH 7.4, 0.1% Triton X 100), plateswere sonicated and lysates spun through 0.2 μm PVDF plates (Corning). 40μL of lysates was assayed for AP activity in alkaline buffer solution(Sigma) containing 1 mg/ml p-Nitrophenyl Phosphate. After incubating for30 min at 37° C., the plates were read on an Envision plate reader at405 nm. Total protein was quantified with a BCA protein assay kit fromPierce according to manufacturer's instructions. AP activity wasnormalized against total protein. Note that “A” indicates that the IC₅₀is less than 20 nM, “B” indicates that the IC₅₀ is 20-100 nM, “C”indicates that the IC₅₀ is >100 nM.

TABLE 1 Approximate EC₅₀ for Inhibition Differentiation Compound AssayEC₅₀ 1 A 11 C 12 B 19 C 31 B 35 C 40 A 41 C 50 C

Example 13 Pancreatic Cancer Model

The activity of Compound 12 was further tested in a human pancreaticmodel: BXPC-3 cells were implanted subcutaneously into the flanks of theright legs of mice. On day 42 post-tumor implant, the mice wererandomized into two groups to receive either Vehicle (30% HPBCD) orCompound 12. Compound 12 was administered orally at 30 mg/kg/day. Afterreceiving 25 daily doses, Compound 12 reduced tumor volume growth by 16%when compared to the vehicle control. At the end of the study, thetumors were harvested 4 hours post the last dose to evaluate an ontarget response by q-RT-PCR analysis of the HH pathway genes. Analysisof human Gli-1 resulted in no modulation. Analysis of murine Gli-1 mRNAlevels resulted in a robust down-regulation in the Compound treatedgroup, when compared to the Vehicle treated group. Inhibition of thehedgehog pathway in mouse cells, but not human tumor cells, indicatesthat one effect of the hedgehog inhibitor is to affect a tumor-stromainteraction.

Example 14 Medulloblastoma Model

The activity of Compound 12 was also evaluated in a transgenic mousemodel of medulloblastoma. Mice that are heterozygous for loss offunction mutations in the tumor suppressors Patched1 (Ptch1) andHypermethylated in Cancer (Hic1) develop spontaneous medulloblastoma.Similar to human medulloblastoma, these tumors demonstrate completepromoter hypermethylation of the remaining Hic1 allele, as well as lossof expression of the wild type Ptch1 allele. When passaged assubcutaneous allografts, these tumors grow aggressively and are Hedgehogpathway-dependent. This model was employed to evaluate the efficacy oforally administered Compound, and to correlate activity with drugexposure in plasma and tumors. Oral administration (PO) of a single doseof Compound 12 led to dose-dependent down-regulation of the HH pathwayin subcutaneously implanted tumors, as measured by decreased Gli-1 mRNAexpression 8 hours post dose administration.

Daily (QD) administration of the Compound PO led to a dose dependentinhibition of tumor growth, with frank tumor regression seen at higherdoses. The approximate effective daily oral dose for 50% inhibition oftumor growth (ED50) is between 3 and 7.5 mg/kg. This demonstrates thatthe hedgehog inhibitor Compound 12 inhibits both the hedgehog pathwayand tumor growth in a tumor dependent on the hedgehog pathway due to agenetic mutation.

Example 15 Multiple Myeloma

The ability of Compound 12 to inhibit the growth of multiple myelomacells (MM) in vitro was tested, using human multiple myeloma cells lines(NCI-H929 and KMS12) and primary clinical bone marrow specimens derivedfrom patients with MM. The cells were treated for 96 hours withCompound, washed, then plated in methylcellulose. Tumor colonies werequantified 10-21 days later as an indicator of cell growth potentialfollowing treatment. Treatment of cell lines or primary patientspecimens resulted in decreased cell growth compared to an untreatedcontrol. Where the untreated control showed 100% growth of cells, eachof the treated cell lines, as well as the clinical samples, showed lessthan about 25% growth.

Example 16 Acute Myeloid Leukemia and Myelodysplastic Syndrome

The ability of Compound 12 to inhibit the in vitro growth of human celllines derived from patients with acute myeloid leukemia (AML, cell lineU937) and myelodysplastic syndrome (MDS, cell line KG1 and KG1a) wasstudied. Each of the cell lines was treated for 72 hours with Compound12 (1.0 uM) followed by plating in methylcellulose. Growth of these celllines was inhibited by Compound 12, as summarized in the table below.

TABLE 2 Inhibition of cell growth in AML and MDS Disease AML MDS Cellline U937 KG1 KG1a % colony formation 43.4 25.1 34.6 with Compound 12(relative to vehicle control)

Example 17 Non-Hodgkin's Lymphoma (NHL) and Hodgkin's Disease (HD)

The ability of Compound 12 to inhibit the in vitro growth of human celllines derived from patients with non-Hodgkin's lymphoma (cell lines RLand Jeko-1) and Hodgkin's disease (cell line L428) was studied. Each ofthe cell lines was treated for 72 hours with Compound 12 (1.0 uM)followed by plating in methylcellulose. Growth of these cell lines wasinhibited by Compound 12, as summarized in the table below.

TABLE 3 Inhibition of cell growth in HD and NHL Disease HD NHL Cell lineL428 RL Jeko-1 % colony formation 21.4 14.3 27.4 with Compound 12(relative to vehicle control)

Example 18 Pre-B Cell Acute Lymphocytic Leukemia

The activity of Compound 12 (1 uM) against three pre-B cell acutelymphocytic leukemia cell lines (REH, RS4-11, and Nalm-6) was studied,using a transient transfection assay in which a Gli-reponsive luciferasereporter was transiently transfected into cells. Treatment with Compound12 repressed luciferase activity compared to a vehicle treated control(Table 4). This demonstrates that Compound 12 is an effective antagonistof the hedgehog pathway.

TABLE 4 Repression of luciferase activity Cell line REH RS4-11 Nalm-6Relative luc activity (vehicle alone) 6.73 12.97 8.42 Relative lucactivity (+ Compound) 1.12 1.31 1.44

The effect of Compound 12 on the growth of two of these cell lines,treated in vitro for 72 hours, was also studied. Following treatment,cells were washed and plated in methylcellulose. There was littleinhibition of colony formation, but subsequent replating of coloniesdemonstrated a significant inhibition of cell growth (Table 5).

TABLE 5 Inhibition of cell growth in ALL Cell line REH RS4-11 % colonyformation with Compound 63 71 (relative to vehicle control) - 1° plating% colony formation with Compound 9 11 (relative to vehicle control) - 2°plating

INCORPORATION BY REFERENCE

All of the U.S. patents and U.S. published patent applications citedherein are hereby incorporated by reference.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

The invention claimed is:
 1. A compound of the formula:

or a pharmaceutically acceptable salt thereof.
 2. A pharmaceuticalcomposition comprising a compound of the formula:

or a pharmaceutically acceptable salt thereof and at least onepharmaceutically acceptable excipient.
 3. A method of treating cancer,which comprises administering to a subject in need thereof atherapeutically effective amount of a compound of the formula:

or a pharmaceutically acceptable salt thereof; wherein the cancer isselected from the group consisting of acute lymphocytic leukemia, acutemyelocytic leukemia, basal cell carcinoma, bile duct carcinoma, bladdercarcinoma, breast cancer, chondrosarcoma, chronic lymphocytic leukemia,chronic myeloid leukemia, colon cancer, esophageal cancer, gastriccancer, gastrointestinal stromal tumor, glioma, hepatocellular cancer,Hodgkin's disease, leukemia, lung cancer, medulloblastoma, melanoma,multiple myeloma, myelodysplastic syndrome, neuroectodermal tumors,non-Hodgkin's type lymphoma, osteogenic sarcoma, ovarian cancer,pancreatic cancer, prostate cancer, sarcoma, and testicular cancer. 4.The method of claim 3, wherein the cancer is selected from the groupconsisting of acute lymphocytic leukemia, basal cell carcinoma, breastcancer, chondrosarcoma, chronic lymphocytic leukemia, chronic myeloidleukemia, colon cancer, esophageal cancer, gastric cancer, glioma,hepatocellular cancer, lung cancer, medulloblastoma, multiple myeloma,non-Hodgkin's type lymphoma, osteogenic sarcoma, ovarian cancer,pancreatic cancer, and prostate cancer.
 5. The method of claim 3,wherein the cancer is pancreatic cancer.
 6. The method of claim 3,wherein the cancer is chondrosarcoma.
 7. The method of claim 3, whereinthe cancer is lung cancer.
 8. The method of claim 7, wherein the lungcancer selected from the group consisting of small cell lung cancer andnon-small cell lung cancer.
 9. The method of claim 3, wherein the canceris basal cell carcinoma.
 10. The method of claim 3, wherein the canceris medulloblastoma.
 11. The method of claim 3, wherein the cancer isovarian cancer.
 12. The method according to claim 3, wherein the canceris osteogenic sarcoma.
 13. The method of claim 3, wherein the cancer isselected from the group consisting of acute lymphocytic leukemia, acutemyelocytic leukemia, chronic lymphocytic leukemia, chronic myeloidleukemia, Hodgkin's disease, leukemia, multiple myeloma, myelodysplasticsyndrome, and non-Hodgkin's type lymphoma.
 14. The method of claim 3,wherein the cancer is selected from the group consisting of acutelymphocytic leukemia, acute myelocytic leukemia, chronic myelocyticleukemia, and chronic lymphocytic leukemia.
 15. The method of claim 3,wherein the compound or pharmaceutically acceptable salt thereof is usedin combination with one or more chemotherapeutic or other anti-canceragent.
 16. The method of claim 15, wherein the other anti-cancer agentis radiation.
 17. The method of claim 3, wherein the compound isadministered locally to a tumor.
 18. The method of claim 3, wherein thecompound is administered systemically.
 19. The method of claim 3,wherein the mode of administration of said compound is inhalation, oral,intravenous, sublingual, ocular, transdermal, rectal, vaginal, topical,intramuscular, intraperitoneal, epidural, subcutaneous, buccal, ornasal.
 20. The method of claim 19, wherein the mode of administration isoral, intravenous, or topical.
 21. A method of inhibiting activation ofa hedgehog pathway in a patient diagnosed with a hyperproliferativedisorder, comprising administering to the patient a compound, or apharmaceutically acceptable salt thereof, as claimed in claim 1 in anamount sufficient to reduce the activation of the hedgehog pathway in acell of the patient.
 22. The method of claim 21, wherein said hyperproliferative disorder is cancer.
 23. A method of antagonizing thehedgehog pathway in a subject, the method comprising administering tothe subject an effective amount of a compound, or a pharmaceuticallyacceptable salt thereof, as claimed in claim 1.